Recombinant botulinum toxins having a soluble C-terminal portion of a heavy chain, an N-terminal portion of a heavy chain and a light chain

ABSTRACT

The present invention includes recombinant proteins derived from  Clostridium botulinum  toxins. In particular, soluble recombinant  Clostridium botulinum  type A, type B and type E toxin proteins are provided. Methods which allow for the isolation of recombinant proteins free of significant endotoxin contamination are provided. The soluble, endotoxin-free recombinant proteins are used as immunogens for the production of vaccines and antitoxins. These vaccines and antitoxins are useful in the treatment of humans and other animals at risk of intoxication with clostridial toxin.

[0001] This application is a Continuation-In-Part of copendingapplication Ser. No. 08/405,496, filed Mar. 16, 1995.

FIELD OF THE INVENTION

[0002] The present invention relates to the isolation of polypeptidesderived from Clostridium botulinum neurotoxins and the use thereof asimmunogens for the production of vaccines, including multivalentvaccines, and antitoxins.

BACKGROUND OF THE INVENTION

[0003] The genus Clostridium is comprised of gram-positive, anaerobic,spore-forming bacilli. The natural habitat of these organisms is theenvironment and the intestinal tracts of humans and other animals.Indeed, clostridia are ubiquitous; they are commonly found in soil,dust, sewage, marine sediments, decaying vegetation, and mud. [See e.g.,P. H. A. Sneath et al., “Clostridium,” Bergey's Manual® of SystematicBacteriology, Vol. 2, pp. 1141-1200, Williams & Wilkins (1986).] Despitethe identification of approximately 100 species of Clostridium, only asmall number have been recognized as etiologic agents of medical andveterinary importance. Nonetheless, these species are associated withvery serious diseases, including botulism, tetanus, anaerobiccellulitis, gas gangrene, bacteremia, pseudomembranous colitis, andclostridial gastroenteritis. Table 1 lists some of the species ofmedical and veterinary importance and the diseases with which they areassociated. As virtually all of these species have been isolated fromfecal samples of apparently healthy persons, some of these isolates maybe transient, rather than permanent residents of the colonic flora.TABLE 1 Clostridium Species Of Medical And Veterinary Importance*Species Disease C. aminovalericum Bacteriuria (pregnant women) C.argentinense Infected wounds; Bacteremia; Botulism; Infections ofamniotic fluid C. baratii Infected war wounds; Peritonitis; Infectiousprocesses of the eye, ear and prostate C. beijerinckikii Infected woundsC. bifermentans Infected wounds; Abscesses; Gas Gangrene; Bacteremia C.botulinum Food poisoning; Botulism (wound, food, infant) C. butyricumUrinary tract, lower respiratory tract, pleural cavity, and abdominalinfections; Infected wounds; Abscesses; Bacteremia C. cadaverisAbscesses; Infected wounds C. carnis Soft tissue infections; BacteremiaC. chauvoei Blackleg C. clostridioforme Abdominal, cervical, scrotal,pleural, and other infections; Septicemia; Peritonitis; Appendicitis C.cochlearium Isolated from human disease processes, but role in diseaseunknown. C. difficile Antimicrobial-associated diarrhea;Pseudomembranous enterocolitis; Bacteremia; Pyogenic infections C.fallax Soft tissue infections C. ghnoii Soft tissue infections C.glycolicum Wound infections; Abscesses; Peritonitis C. hastiformeInfected war wounds; Bacteremia; Abscesses C. histolyticum Infected warwounds; Gas gangrene; Gingival plaque isolate C. indolisGastrointestinal tract infections C. innocuum Gastrointestinal tractinfections; Empyema C. irregulare Penile lesions C. leptum Isolated fromhuman disease processes, but role in disease unknown. C. limosumBacteremia; Peritonitis; Pulmonary infections C. malenominatum Variousinfectious processes C. novyi Infected wounds; Gas gangrene; Blackleg,Big head (ovine); Redwater disease (bovine) C. oroticum Urinary tractinfections; Rectal abscesses C. paraputrificum Bacteremia; Peritonitis;Infected wounds; Appendicitis C. perfringens Gas gangrene; Anaerobiccellulitis; Intra-abdominal abscesses; Soft tissue infections; Foodpoisoning; Necrotizing pneumonia; Empyema; Meningitis; Bacteremia;Uterine Infections; Enteritis necrotans; Lamb dysentery; Struck; OvineEnterotoxemia; C. putrefaciens Bacteriuria (Pregnant women withbacteremia) C. putrificum Abscesses; Infected wounds; Bacteremia C.ramosum Infections of the abdominal cavity, genital tract, lung, andbiliary tract; Bacteremia C. sartagoforme Isolated from human diseaseprocesses, but role in disease unknown. C. septicum Gas gangrene;Bacteremia; Suppurative infections; Necrotizing enterocolitis; Braxy C.sordellii Gas gangrene; Wound infections; Penile lesions; Bacteremia;Abscesses; Abdominal and vaginal infections C. sphenoides Appendicitis;Bacteremia; Bone and soft tissue infections; Intraperitoneal infections;Infected war wounds; Visceral gas gangrene; Renal abscesses C.sporogenes Gas gangrene; Bacteremia; Endocar- ditis; central nervoussystem and pleuropulmonary infections; Penile lesions; Infected warwounds; Other pyogenic infections C. subterminale Bacteremia; Empyema;Biliary tract, soft tissue and bone infections C. symbiosum Liverabscesses; Bacteremia; Infections resulting due to bowel flora C.tertium Gas gangrene; Appendicitis; Brain abscesses; Intestinal tractand soft tissue infections; Infected war wounds; Periodontitis;Bacteremia C. tetani Tetanus; Infected gums and teeth; Cornealulcerations; Mastoid and middle ear infections; Intra- peritonealinfections; Tetanus neonatorum; Postpartum uterine infections; Softtissue infections, especially related to trauma (in- cluding abrasionsand lacerations); Infections related to use of contaminated needles C.thermosaccharolyticum Isolated from human disease pro- cesses, but rolein disease unknown. # 22-23, Star Publishing Co., Belmont, CA (1992); J.Stephen and R. A. Petrowski, “Toxins Which Traverse Membranes andDeregulate # Cells.” in Bacterial Toxins, 2d ed., pp. 66-67, AmericanSociety for Microbiology (1986); R. Berkow and A. J. Fletcher (eds.), #“Bacterial Diseases.” Merck Manual of Diagnosis and Therapy, 16th ed.,pp. 116-126. Merck Research Laboratories, Rahway. N.J. # (1992); and O.H. Sigmund and C. M. Fraser (eds.), “Clostridial Infections.” MerckVeterinary Manual, 5th ed., pp. 396-409, # Merck & Co., Rahway, N. J.(1979).

[0004] In most cases, the pathogenicity of these organisms is related tothe release of powerful exotoxins or highly destructive enzymes. Indeed,several species of the genus Clostridium produce toxins and otherenzymes of great medical and veterinary significance. [C. L. Hatheway,Clin. Microbiol. Rev. 3:66-98 (1990).]

[0005] Perhaps because of their significance for human and veterinarymedicine, much research has been conducted on these toxins, inparticular those of C. botulinum and C. difficile.

[0006]C. botulinum

[0007] Several strains of Clostridium botulinum produce toxins ofsignificance to human and animal health. [C. L. Hatheway, Clin.Microbiol. Rev. 3:66-98 (1990)] The effects of these toxins range fromdiarrheal diseases that can cause destruction of the colon, to paralyticeffects that can cause death. Particularly at risk for developingclostridial diseases are neonates and humans and animals in poor health(e.g., those suffering from diseases associated with old age orimmunodeficiency diseases).

[0008]Clostridium botulinum produces the most poisonous biological toxinknown. The lethal human dose is a mere 10⁻⁹ mg/kg bodyweight for toxinin the bloodstream. Botulinal toxin blocks nerve transmission to themuscles, resulting in flaccid paralysis. When the toxin reaches airwayand respiratory muscles, it results in respiratory failure that cancause death. [S. Arnon, J. Infect. Dis. 154:201-206 (1986)]

[0009]C. botulinum spores are carried by dust and are found onvegetables taken from the soil, on fresh fruits, and on agriculturalproducts such as honey. Under conditions favorable to the organism, thespores germinate to vegetative cells which produces toxin. [S. Arnon,Ann. Rev. Med. 31:541 (1980)]

[0010] Botulism disease may be grouped into four types, based on themethod of introduction of toxin into the bloodstream. Food-bornebotulism results from ingesting improperly preserved and inadequatelyheated food that contains botulinal toxin. There were 355 cases offood-borne botulism in the United States between 1976 and 1984. [K. L.MacDonald et al., Am. J. Epidemiol. 124:794 (1986).] The death rate dueto botulinal toxin is 12% and can be higher in particular risk groups.[C. O. Tacket et al., Am. J. Med. 76:794 (1984).] Wound-induced botulismresults from C. botulinum penetrating traumatized tissue and producingtoxin that is absorbed into the bloodstream. Since 1950, thirty cases ofwound botulism have been reported. [M. N. Swartz, “AnaerobicSpore-Forming Bacilli: The Clostridia,” pp. 633-646, in B. D. Davis etal., (eds.), Microbiology, 4th edition, J. B. Lippincott Co. (1990).]Inhalation botulism results when the toxin is inhaled. Inhalationbotulism has been reported as the result of accidental exposure in thelaboratory [E. Holzer, Med. Klin. 41:1735 (1962)] and could arise if thetoxin is used as an agent of biological warfare [D. R. Franz et al., inBotulinum and Tetanus Neurotoxins, B. R. DasGupta, ed., Plenum Press,New York (1993), pp. 473-476]. Infectious infant botulism results fromC. botulinum colonization of the infant intestine with production oftoxin and its absorption into the bloodstream. It is likely that thebacterium gains entry when spores are ingested and subsequentlygerminate. [S. Arnon, J. Infect. Dis. 154:201 (1986).] There have been500 cases reported since it was first recognized in 1976. [M. N. Swartz,supra.]

[0011] Infant botulism strikes infants who are three weeks to elevenmonths old (greater than 90% of the cases are infants less than sixmonths). [S. Arnon, J. Infect. Dis. 154:201 (1986).] It is believed thatinfants are susceptible, due, in large part, to the absence of the fulladult complement of intestinal microflora. The benign microflora presentin the adult intestine provide an acidic environment that is notfavorable to colonization by C. botulinum. Infants begin life with asterile intestine which is gradually colonized by microflora. Because ofthe limited microflora present in early infancy, the intestinalenvironment is not as acidic, allowing for C. botulinum sporegermination, growth, and toxin production. In this regard, some adultswho have undergone antibiotic therapy which alters intestinal microflorabecome more susceptible to botulism.

[0012] An additional factor accounting for infant susceptibility toinfectious botulism is the immaturity of the infant immune system. Themature immune system is sensitized to bacterial antigens and producesprotective antibodies. Secretory IgA produced in the adult intestine hasthe ability to agglutinate vegetative cells of C. botulinum. [S. Arnon,J. Infect. Dis. 154:201 (1986).] Secretory IgA may also act bypreventing intestinal bacteria and their products from crossing thecells of the intestine. [S. Arnon, Epidemiol. Rev. 3:45 (1981).] Theinfant immune system is not primed to do this.

[0013] Clinical symptoms of infant botulism range from mild paralysis,to moderate and severe paralysis requiring hospitalization, to fulminantparalysis, leading to sudden death. [S. Arnon, Epidemiol. Rev. 3:45(1981).]

[0014] The chief therapy for severe infant botulism is ventilatoryassistance using a mechanical respirator and concurrent elimination oftoxin and bacteria using cathartics, enemas, and gastric lavage. Therewere 68 hospitalizations in California for infant botulism in a singleyear with a total cost of over $4 million for treatment. [T. L.Frankovich and S. Arnon, West. J. Med. 154:103 (1991).]

[0015] Different strains of Clostridium botulinum each produceantigenically distinct toxin designated by the letters A-G. Serotype Atoxin has been implicated in 26% of the cases of food botulism; types B,E and F have also been implicated in a smaller percentage of the foodbotulism cases [H. Sugiyama, Microbiol. Rev. 44:419 (1980)]. Woundbotulism has been reportedly caused by only types A or B toxins [H.Sugiyama, supra]. Nearly all cases of infant botulism have been causedby bacteria producing either type A or type B toxin. (Exceptionally, oneNew Mexico case was caused by Clostridium botulinum producing type Ftoxin and another by Clostridium botulinum producing a type B-type Fhybrid.) [S. Arnon, Epidemiol. Rev. 3:45 (1981).] Type C toxin affectswaterfowl, cattle, horses and mink. Type D toxin affects cattle, andtype E toxin affects both humans and birds.

[0016] A trivalent antitoxin derived from horse plasma is commerciallyavailable from Connaught Industries Ltd. as a therapy for toxin types A,B, and E. However, the antitoxin has several disadvantages. First,extremely large dosages must be injected intravenously and/orintramuscularly. Second, the antitoxin has serious side effects such asacute anaphylaxis which can lead to death, and serum sickness. Finally,the efficacy of the antitoxin is uncertain and the treatment is costly.[C. O. Tacket et al., Am. J. Med. 76:794 (1984).]

[0017] A heptavalent equine botulinal antitoxin which uses only theF(ab′)₂ portion of the antibody molecule has been tested by the UnitedStates Military. [M. Balady, USAMRDC Newsletter, p. 6 (1991).] This wasraised against impure toxoids in those large animals and is not a hightiter preparation.

[0018] A pentavalent human antitoxin has been collected from immunizedhuman subjects for use as a treatment for infant botulism. The supply ofthis antitoxin is limited and cannot be expected to meet the needs ofall individuals stricken with botulism disease. In addition, collectionof human sera must involve screening out HIV and other potentiallyserious human pathogens. [P. J. Schwarz and S. S. Arnon, Western J. Med.156:197 (1992).]

[0019] Infant botulism has been implicated as the cause of mortality insome cases of Sudden Infant Death Syndrome (SIDS, also known as cribdeath). SIDS is officially recognized as infant death that is sudden andunexpected and that remained unexplained despite complete post-mortemexamination. The link of SIDS to infant botulism came when fecal orblood specimens taken at autopsy from SIDS infants were found to containC. botulinum organisms and/or toxin in 3-4% of cases analyzed. [D. R.Peterson et al., Rev. Infect. Dis. 1:630 (1979).] In contrast, only 1 of160 healthy infants (0.6%) had C. botulinum organisms in the feces andno botulinal toxin. (S. Arnon et al., Lancet, pp. 1273-76, Jun. 17,1978.)

[0020] In developed countries, SIDS is the number one cause of death inchildren between one month and one year old. (S. Arnon et al., Lancet,pp. 1273-77, Jun. 17, 1978.) More children die from SIDS in the firstyear than from any other single cause of death in the first fourteenyears of life. In the United States, there are 8,000-10,000 SIDS victimsannually. Id.

[0021] What is needed is an effective therapy against infant botulismthat is free of dangerous side effects, is available in large supply ata reasonable price, and can be safely and gently delivered so thatprophylactic application to infants is feasible.

[0022] Immunization of subjects with toxin preparations has been done inan attempt to induce immunity against botulinal toxins. A C. botulinumvaccine comprising chemically inactivated (i.e., formaldehyde-treated)type A, B, C, D and E toxin is commercially available for human usage.However, this vaccine preparation has several disadvantages. First, theefficacy of this vaccine is variable (in particular, only 78% ofrecipients produce protective levels of anti-type B antibodies followingadministration of the primary series). Second, immunization is painful(deep subcutaneous inoculation is required for administration), withadverse reactions being common (moderate to severe local reactions occurin approximately 6% of recipients upon initial injection; this numberrises to approximately 11% of individuals who receive boosterinjections) [Informational Brochure for the Pentavalent (ABCDE)Botulinum Toxoid, Centers for Disease Control]. Third, preparation ofthe vaccine is dangerous as active toxin must be handled by laboratoryworkers.

[0023] What is needed are safe and effective vaccine preparations foradministration to those at risk of exposure to C. botulinum toxins.

[0024]C. difficile

[0025]C. difficile, an organism which gained its name due todifficulties encountered in its isolation, has recently been proven tobe an etiologic agent of diarrheal disease. (Sneath et al, p. 1165.). C.difficile is present in the gastrointestinal tract of approximately 3%of healthy adults, and 10-30% of neonates without adverse effect(Swartz, at p. 644); by other estimates, C. difficile is a part of thenormal gastrointestinal flora of 2-10% of humans. [G. F. Brooks et al.,(eds.) “Infections Caused by Anaerobic Bacteria,” Jawetz, Melnick, &Adelberg's Medical Microbiology, 19th ed., pp. 257-262, Appleton &Lange, San Mateo, Calif. (1991).] As these organisms are relativelyresistant to most commonly used antimicrobials, when a patient istreated with antibiotics, the other members of the normalgastrointestinal flora are suppressed and C. difficile flourishes,producing cytopathic toxins and enterotoxins. It has been found in 25%of cases of moderate diarrhea resulting from treatment with antibiotics,especially the cephalosporins, clindamycin, and ampicillin. [M. N.Swartz at 644.]

[0026] Importantly, C. difficile is commonly associated with nosocomialinfections. The organism is often present in the hospital and nursinghome environments and may be carried on the hands and clothing ofhospital personnel who care for debilitated and immunocompromisedpatients. As many of these patients are being treated withantimicrobials or other chemotherapeutic agents, such transmission of C.difficile represents a significant risk factor for disease. (Engelkirket al., pp. 64-67.)

[0027]C. difficile is associated with a range of diarrhetic illness,ranging from diarrhea alone to marked diarrhea and necrosis of thegastrointestinal mucosa with the accumulation of inflammatory cells andfibrin, which forms a pseudomembrane in the affected area. (Brooks etal) It has been found in over 95% of pseudomembranous enterocolitiscases. (Swartz, at p. 644.) This occasionally fatal disease ischaracterized by diarrhea, multiple small colonic plaques, and toxicmegacolon. (Swartz, at p. 644.) Although stool cultures are sometimesused for diagnosis, diagnosis is best made by detection of the heatlabile toxins present in fecal filtrates from patients withenterocolitis due to C. difficile. (Swartz, at p. 644-645; and Brooks etal., at p. 260.) C. difficile toxins are cytotoxic for tissue/cellcultures and cause enterocolitis when injected intracecally intohamsters. (Swartz, at p. 644.)

[0028] The enterotoxicity of C. difficile is primarily due to the actionof two toxins, designated A and B, each of approximately 300,000 inmolecular weight. Both are potent cytotoxins, with toxin A possessingdirect enterocytotoxic activity. [Lyerly et al., Infect. Immun. 60:4633(1992).] Unlike toxin A of C. perfringens, an organism rarely associatedwith antimicrobial-associated diarrhea, the toxin of C. difficile is nota spore coat constituent and is not produced during sporulation.(Swartz, at p. 644.) C. difficile toxin A causes hemorrhage, fluidaccumulation and mucosal damage in rabbit ileal loops and appears toincrease the uptake of toxin B by the intestinal mucosa. Toxin B doesnot cause intestinal fluid accumulation, but it is 1000 times more toxicthan toxin A to tissue culture cells and causes membrane damage.Although both toxins induce similar cellular effects such as actindisaggregation, differences in cell specificity occurs.

[0029] Both toxins are important in disease. [Borriello et al., Rev.Infect. Dis., 12(suppl. 2):S185 (1990); Lyerly et al., Infect. Immun.,47:349 (1985); and Rolfe, Infect. Immun., 59:1223 (1990).] Toxin A isthought to act first by binding to brush border receptors, destroyingthe outer mucosal layer, then allowing toxin B to gain access to theunderlying tissue. These steps in pathogenesis would indicate that theproduction of neutralizing antibodies against toxin A may be sufficientin the prophylactic therapy of CDAD. However, antibodies against toxin Bmay be a necessary additional component for an effective therapeuticagainst later stage colonic disease. Indeed, it has been reported thatanimals require antibodies to both toxin A and toxin B to be completelyprotected against the disease. [Kim and Rolfe, Abstr. Ann. Meet. Am.Soc. Microbiol., 69:62 (1987).]

[0030]C. difficile has also been reported to produce other toxins suchas an enterotoxin different from toxins A and B [Banno et al., Rev.Infect. Dis., 6(Suppl. 1:S11-S20 (1984)], a low molecular weight toxin[Rihn et al., Biochem. Biophys. Res. Comm., 124:690-695 (1984)], amotility altering factor [Justus et al, Gastroenterol., 83:836-843(1982)], and perhaps other toxins. Regardless, C. difficilegastrointestinal disease is of primary concern.

[0031] It is significant that due to its resistance to most commonlyused antimicrobials, C. difficile is associated with antimicrobialtherapy with virtually all antimicrobial agents (although most commonlyampicillin, clindamycin and cephalosporins). It is also associated withdisease in patients undergoing chemotherapy with such compounds asmethotrexate, 5-fluorouracil, cyclophosphamide, and doxorubicin. [S. M.Finegold et al., Clinical Guide to Anaerobic Infections, pp. 88-89, StarPublishing Co., Belmont, Calif. (1992).]

[0032] Treatment of C. difficile disease is problematic, given the highresistance of the organism. Oral metronidazole, bacitracin andvancomycin have been reported to be effective. (Finegold et al., p. 89.)However there are problems associated with treatment utilizing thesecompounds. Vancomycin is very expensive, some patients are unable totake oral medication, and the relapse rate is high (20-25%), although itmay not occur for several weeks. Id.

[0033]C. difficile disease would be prevented or treated by neutralizingthe effects of these toxins in the gastrointestinal tract. Thus, what isneeded is an effective therapy against C. difficile toxin that is freeof dangerous side effects, is available in large supply at a reasonableprice, and can be safely delivered so that prophylactic application topatients at risk of developing pseudomembranous enterocolitis can beeffectively treated.

DESCRIPTION OF THE DRAWINGS

[0034]FIG. 1 shows the reactivity of anti-C. botulinum IgY by Westernblot.

[0035]FIG. 2 shows the IgY antibody titer to C. botulinum type A toxoidin eggs, measured by ELISA.

[0036]FIG. 3 shows the results of C. difficile toxin A neutralizationassays.

[0037]FIG. 4 shows the results of C. difficile toxin B neutralizationassays.

[0038]FIG. 5 shows the results of C. difficile toxin B neutralizationassays.

[0039]FIG. 6 is a restriction map of C. difficile toxin A gene, showingsequences of primers 1-4 (SEQ ID NOS:1-4).

[0040]FIG. 7 is a Western blot of C. difficile toxin A reactive protein.

[0041]FIG. 8 shows C. difficile toxin A expression constructs.

[0042]FIG. 9 shows C. difficile toxin A expression constructs.

[0043]FIG. 10 shows the purification of recombinant C. difficile toxinA.

[0044]FIG. 11 shows the results of C. difficile toxin A neutralizationassays with antibodies reactive to recombinant toxin A.

[0045]FIG. 12 shows the results for a C. difficile toxin Aneutralization plate.

[0046]FIG. 13 shows the results for a C. difficile toxin Aneutralization plate.

[0047]FIG. 14 shows the results of recombinant C. difficile toxin Aneutralization assays.

[0048]FIG. 15 shows C. difficile toxin A expression constructs.

[0049]FIG. 16 shows a chromatograph plotting absorbance at 280 nmagainst retention time for a pMA1870-680 IgY PEG preparation.

[0050]FIG. 17 shows two recombinant C. difficile toxin B expressionconstructs.

[0051]FIG. 18 shows C. difficile toxin B expression constructs.

[0052]FIG. 19 shows C. difficile toxin B expression constructs.

[0053]FIG. 20 shows C. difficile toxin B expression constructs.

[0054]FIG. 21 is an SDS-PAGE gel showing the purification of recombinantC. difficile toxin B fusion protein.

[0055]FIG. 22 is an SDS-PAGE gel showing the purification of twohistidine-tagged recombinant C. difficile toxin B proteins.

[0056]FIG. 23 shows C. difficile toxin B expression constructs.

[0057]FIG. 24 is a Western blot of C. difficile toxin B reactiveprotein.

[0058]FIG. 25 shows C. botulinum type A toxin expression constructs;constructs used to provide C. botulinum or C. difficile sequences arealso shown.

[0059]FIG. 26 is an SDS-PAGE gel stained with Coomaisse blue showing thepurification of recombinant C. botulinum type A toxin fusion proteins.

[0060]FIG. 27 shows C. botulinum type A toxin expression constructs;constructs used to provide C. botulinum sequences are also shown.

[0061]FIG. 28 is an SDS-PAGE gel stained with Coomaisse blue showing thepurification of pHisBot protein using the Ni-NTA resin.

[0062]FIG. 29 is an SDS-PAGE gel stained with Coomaisse blue showing theexpression of pHisBot protein in BL21(DE3) and BL21(DE3)pLysS hostcells.

[0063]FIG. 30 is an SDS-PAGE gel stained with Coomaisse blue showing thepurification of pHisBot protein using a batch absorption procedure.

[0064]FIG. 31 is an SDS-PAGE gel stained with Coomaisse blue showing thepurification of pHisBot and pHisBot(native) proteins using a Ni-NTAcolumn.

[0065]FIG. 32 is an SDS-PAGE gel stained with Coomaisse blue showing thepurification of pHisBotA protein expressed in pHisBotA(syn) kan lacIqT7/pACYCGro/BL21(DE3) cells using an IDA column.

[0066]FIG. 33 is an SDS-PAGE gel stained with Coomaisse blue showing thepurification of pHisBotA, pHisBotB and pHisBotE proteins by IDAchromatography followed by chromatography on S-100 to remove foldingchaperones.

[0067]FIG. 34 is an SDS-PAGE gel stained with Coomaisse blue showing theextracts derived from pHisBotB amp T7lac/BL21(DE3) cells before andafter purification on a Ni-NTA column.

[0068]FIG. 35 is an SDS-PAGE gel run under native conditions and stainedwith Coomaisse blue showing the removal of folding chaperones fromIDA-purified BotB protein using a S-100 column.

[0069]FIG. 36 is an SDS-PAGE gel stained with Coomaisse blue showingproteins that eluted during an imidazole step gradient applied to a IDAcolumn containing a lysate of pHisBotB kan lacIq T7/pACYCGro/BL21(DE3)cells.

[0070]FIG. 37 is an SDS-PAGE gel run under native conditions and stainedwith Coomaisse blue showing IDA-purified BotB protein before and afterultrafiltration.

[0071]FIG. 38 is an SDS-PAGE gel stained with Coomaisse blue showing thepurification of BotE protein using a NiNTA column.

[0072]FIG. 39 is an SDS-PAGE gel stained with Coomaisse blue showingextracts derived from pHisBotA kan T7 lac/BL21(DE3) pLysS cells grown infermentation culture.

[0073]FIG. 40 is a chromatogram showing proteins present afterIDA-purified BotE protein was applied to a S-100 column.

DEFINITIONS

[0074] To facilitate understanding of the invention, a number of termsare defined below.

[0075] As used herein, the term “neutralizing” is used in reference toantitoxins, particularly antitoxins comprising antibodies, which havethe ability to prevent the pathological actions of the toxin againstwhich the antitoxin is directed.

[0076] As used herein, the term “overproducing” is used in reference tothe production of clostridial toxin polypeptides in a host cell andindicates that the host cell is producing more of the clostridial toxinby virtue of the introduction of nucleic acid sequences encoding saidclostridial toxin polypeptide than would be expressed by said host cellabsent the introduction of said nucleic acid sequences. To allow ease ofpurification of toxin polypeptides produced in a host cell it ispreferred that the host cell express or overproduce said toxinpolypeptide at a level greater than 1 mg/liter of host cell culture.

[0077] “A host cell capable of expressing a recombinant protein at alevel greater than or equal to 5% of the total cellular protein” is ahost cell in which the recombinant protein represents at least 5% of thetotal cellular protein. To determine what percentage of total cellularprotein the recombinant protein represents, the following steps aretaken. A total of 10 OD₆₀₀ units of recombinant host cells (e.g., 200 μlof cells at OD₆₀₀=50/ml) are removed (at a timepoint known to representthe peak of expression of the desired recombinant protein) to a 1.5 mlmicrofuge tube and pelleted for 2 min at maximum rpm in a microfuge. Thepellets are resuspended in 1 ml of 50 mM NaHPO₄, 0.5 M NaCl, 40 mMimidazole buffer (pH 6.8) containing 1 mg/ml lysozyme. The samples areincubated for 20 min at room temperature and stored ON at −70° C.Samples are thawed completely at room temperature and sonicated 2×10seconds with a Branson Sonifier 450 microtip probe at # 3 power setting.The samples are centrifuged for 5 min. at maximum rpm in a microfuge. Analiquot (20 μl) of the protein sample is removed to 20 μl 2× samplebuffer (this represents the total protein extract). The samples areheated to 95° C. for 5 min, then cooled and 5 or 10 μl are loaded onto12.5% SDS-PAGE gels. High molecular weight protein markers are alsoloaded to allow for estimation of the MW of identified recombinantproteins. After electrophoresis, protein is detected generally bystaining with Coomassie blue and the stained gel is scanned using adensitometer to determine the percentage of protein present in eachband. In this manner, the percentage of protein present in the bandcorresponding to the recombinant protein of interest may be determined.It is not necessary that Coomassie blue be employed for the detection ofprotein, a number of fluorescent dyes [e.g., Sypro orange S-6651(Molecular Probes, Eugene, Oreg.] may be employed and the stained gelscanned using a fluoroimager [e.g., Fluor Imager SI (Molecular Dynamics,Sunnyvale, Calif.)].

[0078] “A host cell capable of expressing a recombinant protein as asoluble protein at a level greater than or equal to 0.25% of the totalsoluble cellular protein” is a host cell in which the amount of solublerecombinant protein present represents at least 0.25% of the totalcellular protein. As used herein “total soluble cellular protein” refersto a clarified PEI lysate prepared as described in Example 31(c)(iv).Briefly, cells are harvested following induction of expression ofrecombinant protein (at a point of maximal expression). The cells areresuspended in cell resuspension buffer (CRB: 50 mM NAPO₄, 0.5 M NaCl,40 mM imidazole, pH 6.8) to create a 20% cell suspension (wet weight ofcells/volume of CRB) and cell lysates are prepared as described inExample 31(c)(iv) (i.e., sonication or homogenization followed bycentrifugation). The cell lysate is then flocculated utilizingpolyethyleneimine (PEI) prior to centrifugation. PEI (a 2% solution indH₂O, pH 7.5 with HCl) is added to the cell lysate to a finalconcentration of 0.2%, and stirred for 20 min at room temperature priorto centrifugation [8,500 rpm in JA10 rotor (Beckman) for 30 minutes at4° C.]. This treatment removes RNA, DNA and cell wall components,resulting in a clarified, low viscosity lysate (“PEI clarified lysate”)The recombinant protein present in the PEI clarified lysate is thenpurified (e.g., by chromatography on an IDA column for his-taggedproteins). The amount of purified recombinant protein (i.e., the elutedprotein) is divided by the concentration of protein present in the PEIclarified lysate (typically 8 mg/ml when using a 20% cell suspension asthe starting material) and multiplied by 100 to determine whatpercentage of total soluble cellular protein is comprised of the solublerecombinant protein (see Example 33b).

[0079] As used herein, the term “fusion protein” refers to a chimericprotein containing the protein of interest (i.e., C. botulinum toxin A,B, C, D, E, F, or G and fragments thereof) joined to an exogenousprotein fragment (the fusion partner which consists of a non-toxinprotein). The fusion partner may enhance solubility of the C. botulinumprotein as expressed in a host cell, may provide an affinity tag toallow purification of the recombinant fusion protein from the host cellor culture supernatant, or both. If desired, the fusion protein may beremoved from the protein of interest (i.e., toxin protein or fragmentsthereof) prior to immunization by a variety of enzymatic or chemicalmeans known to the art.

[0080] As used herein the term “non-toxin protein” or “non-toxin proteinsequence” refers to that portion of a fusion protein which comprises aprotein or protein sequence which is not derived from a bacterial toxinprotein.

[0081] The term “protein of interest” as used herein refers to theprotein whose expression is desired within the fusion protein. In afusion protein the protein of interest will be joined or fused withanother protein or protein domain, the fusion partner, to allow forenhanced stability of the protein of interest and/or ease ofpurification of the fusion protein.

[0082] As used herein, the term “maltose binding protein” refers to themaltose binding protein of E. coli. A portion of the maltose bindingprotein may be added to a protein of interest to generate a fusionprotein; a portion of the maltose binding protein may merely enhance thesolubility of the resulting fusion protein when expressed in a bacterialhost. On the other hand, a portion of the maltose binding protein mayallow affinity purification of the fusion protein on an amylose resin.

[0083] As used herein, the term “poly-histidine tract” when used inreference to a fusion protein refers to the presence of two to tenhistidine residues at either the amino- or carboxy-terminus of a proteinof interest. A poly-histidine tract of six to ten residues is preferred.The poly-histidine tract is also defined functionally as being a numberof consecutive histidine residues added to the protein of interest whichallows the affinity purification of the resulting fusion protein on anickel-chelate or IDA column.

[0084] As used herein, the term “purified” or “to purify” refers to theremoval of contaminants from a sample. For example, antitoxins arepurified by removal of contaminating non-immunoglobulin proteins; theyare also purified by the removal of immunoglobulin that does not bindtoxin. The removal of non-immunoglobulin proteins and/or the removal ofimmunoglobulins that do not bind toxin results in an increase in thepercent of toxin-reactive immunoglobulins in the sample. In anotherexample, recombinant toxin polypeptides are expressed in bacterial hostcells and the toxin polypeptides are purified by the removal of hostcell proteins; the percent of recombinant toxin polypeptides is therebyincreased in the sample. Additionally, the recombinant toxinpolypeptides are purified by the removal of host cell components such aslipopolysaccharide (e.g., endotoxin).

[0085] The term “recombinant DNA molecule” as used herein refers to aDNA molecule which is comprised of segments of DNA joined together bymeans of molecular biological techniques.

[0086] The term “recombinant protein” or “recombinant polypeptide” asused herein refers to a protein molecule which is expressed from arecombinant DNA molecule.

[0087] The term “native protein” as used herein refers to a proteinwhich is isolated from a natural source as opposed to the production ofa protein by recombinant means.

[0088] As used herein the term “portion” when in reference to a protein(as in “a portion of a given protein”) refers to fragments of thatprotein. The fragments may range in size from four amino acid residuesto the entire amino acid sequence minus one amino acid.

[0089] As used herein “soluble” when in reference to a protein producedby recombinant DNA technology in a host cell is a protein which existsin solution in the cytoplasm of the host cell; if the protein contains asignal sequence the soluble protein is exported to the periplasmic spacein bacteria hosts and is secreted into the culture medium in eucaryoticcells capable of secretion or by bacterial host possessing theappropriate genes (i.e., the kil gene). In contrast, an insolubleprotein is one which exists in denatured form inside cytoplasmicgranules (called inclusion bodies) in the host cell. High levelexpression (i.e., greater than 10-20 mg recombinant protein/liter ofbacterial culture) of recombinant proteins often results in theexpressed protein being found in inclusion bodies in the bacterial hostcells. A soluble protein is a protein which is not found in an inclusionbody inside the host cell or is found both in the cytoplasm and ininclusion bodies and in this case the protein may be present at high orlow levels in the cytoplasm.

[0090] A distinction is drawn between a soluble protein (i.e., a proteinwhich when expressed in a host cell is produced in a soluble form) and a“solubilized” protein. An insoluble recombinant protein found inside aninclusion body may be solubilized (i.e., rendered into a soluble form)by treating purified inclusion bodies with denaturants such as guanidinehydrochloride, urea or sodium dodecyl sulfate (SDS). These denaturantsmust then be removed from the solubilized protein preparation to allowthe recovered protein to renature (refold). Not all proteins will refoldinto an active conformation after solubilization in a denaturant andremoval of the denaturant. Many proteins precipitate upon removal of thedenaturant. SDS may be used to solubilize inclusion bodies and willmaintain the proteins in solution at low concentration. However,dialysis will not always remove all of the SDS (SDS can form micelleswhich do not dialyze out); therefore, SDS-solubilized inclusion bodyprotein is soluble but not refolded.

[0091] A distinction is drawn between proteins which are soluble (i.e.,dissolved) in a solution devoid of significant amounts of ionicdetergents (e.g., SDS) or denaturants (e.g., urea, guanidinehydrochloride) and proteins which exist as a suspension of insolubleprotein molecules dispersed within the solution. A soluble protein willnot be removed from a solution containing the protein by centrifugationusing conditions sufficient to remove bacteria present in a liquidmedium (i.e., centrifugation at 12,000×g for 4-5 minutes). For example,to test whether two proteins, protein A and protein B, are soluble insolution, the two proteins are placed into a solution selected from thegroup consisting of PBS-NaCl (PBS containing 0.5 M NaCl), PBS-NaClcontaining 0.2% Tween 20, PBS, PBS containing 0.2% Tween 20, PBS-C (PBScontaining 2 mM CaCl₂), PBS-C containing either 0.1 or 0.5% Tween 20,PBS-C containing either 0.1 or 0.5% NP-40, PBS-C containing either 0.1or 0.5% Triton X-100, PBS-C containing 0.1% sodium deoxycholate. Themixture containing proteins A and B is then centrifuged at 5000×g for 5minutes. The supernatant and pellet formed by centrifugation are thenassayed for the presence of protein A and B. If protein A is found inthe supernatant and not in the pellet [except for minor amounts (i.e.,less than 10%) as a result of trapping], protein is said to be solublein the solution tested. If the majority of protein B is found in thepellet (i.e., greater than 90%), then protein B is said to exist as asuspension in the solution tested.

[0092] As used herein, the term “therapeutic amount” refers to thatamount of antitoxin required to neutralize the pathologic effects of oneor more clostridial toxins in a subject.

[0093] The term “pyrogen” as used herein refers to a fever-producingsubstance. Pyrogens may be endogenous to the host (e.g., prostaglandins)or may be exogenous compounds (e.g., bacterial endo- and exotoxins,nonbacterial compounds such as antigens and certain steroid compounds,etc.). The presence of pyrogen in a pharmaceutical solution may bedetected using the U.S. Pharmacopeia (USP) rabbit fever test (UnitedStates Pharmacopeia, Vol. XXII (1990) United States PharmacopeialConvention, Rockville, Md., p. 151).

[0094] The term “endotoxin” as used herein refers to the high molecularweight complexes associated with the outer membrane of gram-negativebacteria Unpurified endotoxin contains lipids, proteins andcarbohydrates. Highly purified endotoxin does not contain protein and isreferred to as lipopolysaccharide (LPS). Because unpurified endotoxin isof concern in the production of pharmaceutical compounds (e.g., proteinsproduced in E. coli using recombinant DNA technology), the termendotoxin as used herein refers to unpurified endotoxin. Bacterialendotoxin is a well known pyrogen.

[0095] As used herein, the term “endotoxin-free” when used in referenceto a composition to be administered parenterally (with the exception ofintrathecal administration) to a host means that the dose to bedelivered contains less than 5 EU/kg body weight [FDA Guidelines forParenteral Drugs (December 1987)]. Assuming a weight of 70 kg for anadult human, the dose must contain less than 350 EU to meet FDAGuidelines for parenteral administration. Endotoxin levels are measuredherein using the Limulus Amebocyte Lysate (LAL) test (Limulus AmebocyteLysate Pyrochrome™, Associates of Cape Cod, Inc. Woods Hole, Mass.). Tomeasure endotoxin levels in preparations of recombinant proteins, 0.5 mlof a solution comprising 0.5 mg of purified recombinant protein in 50 mMNaPO₄, pH 7.0, 0.3M NaCl and 10% glycerol is used in the LAL assayaccording to the manufacturer's instructions for the endpointchromogenic without diazo-coupling method [the specific components ofthe buffer containing recombinant protein to be analyzed in the LAL testare not important; any buffer having a neutral pH may be employed (seefor example, alternative buffers employed in Examples 34, 40 and 45)].Compositions containing less than or equal to than 250 endotoxin units(EU)/mg of purified recombinant protein are herein defined as“substantially endotoxin-free.” Preferably the composition contains lessthan or equal to 100, and most preferably less than or equal to 60,(EU)/mg of purified recombinant protein. Typically, administration ofbacterial toxins or toxoids to adult humans for the purpose ofvaccination involves doses of about 10-500 μg protein/dose. Therefore,administration of 10-500 μg of a purified recombinant protein to a 70 kghuman, wherein said purified recombinant protein preparation contains 60EU/mg protein, results in the introduction of only 0.6 to 30 EU (i.e.,0.2 to 8.6% of the maximum allowable endotoxin burden per parenteraldose). Administration of 10-500 μg of a purified recombinant protein toa 70 kg human, wherein said purified recombinant protein preparationcontains 250 EU/mg protein, results in the introduction of only 2.5 to125 EU (i.e., 0.7 to 36% of the maximum allowable endotoxin burden perparenteral dose).

[0096] The LAL test is accepted by the U.S. FDA as a means of detectingbacterial endotoxins (21 C.F.R. §§ 660.100-105). Studies have shown thatthe LAL test is equivalent or superior to the USP rabbit pyrogen testfor the detection of endotoxin and thus the LAL test can be used as asurrogate for pyrogenicity studies in animals [F. C. Perason, Pyrogens:endotoxins, LAL testing and depyrogenation, Marcel Dekker, New York(1985), pp.150-155]. The FDA Bureau of Biologics accepts the LAL assayin place of the USP rabbit pyrogen test so long as the LAL assayutilized is shown to be as sensitive as, or more sensitive as the rabbittest [Fed. Reg., 38, 26130 (1980)].

[0097] The term “monovalent” when used in reference to a clostridialvaccine refers to a vaccine which is capable of provoking an immuneresponse in a host animal directed against a single type of clostridialtoxin. For example, if immunization of a host with C. botulinum type Atoxin vaccine induces antibodies in the immunized host which protectagainst a challenge with type A toxin but not against challenge withtype B, C, D, E, F or G toxins, then the type A vaccine is said to bemonovalent. In contrast, a “multivalent” vaccine provokes an immuneresponse in a host animal directed against several (i.e., more than one)clostridial toxins. For example, if immunization of a host with avaccine comprising C. botulinum type A and B toxins induces theproduction of antibodies which protect the host against a challenge withboth type A and B toxin, the vaccine is said to be multivalent (inparticular, this hypothetical vaccine is bivalent).

[0098] As used herein the term “immunogenically-effective amount” refersto that amount of an immunogen required to invoke the production ofprotective levels of antibodies in a host upon vaccination.

[0099] The term “protective level”, when used in reference to the levelof antibodies induced upon immunization of the host with an immunogenwhich comprises a bacterial toxin, means a level of circulatingantibodies sufficient to protect the host from challenge with a lethaldose of the toxin.

[0100] As used herein the terms “protein” and “polypeptide” refer tocompounds comprising amino acids joined via peptide bonds and are usedinterchangeably.

[0101] The terms “toxin” and “neurotoxin” when used in reference totoxins produced by members (i.e., species and strains) of the genusClostridium are used interchangeably and refer to the proteins which arepoisonous to nerve tissue.

[0102] The term “receptor-binding domain” when used in reference to a C.botulinum toxin refers to the carboxy-terminal portion of the heavychain (H_(C) or the C fragment) of the toxin which is presumed to beresponsible for the binding of the active toxin (i.e., the derivativetoxin comprising the H and L chains joined via disulfide bonds) toreceptors on the surface of synaptosomes. The receptor-binding domainfor C. botulinum type A toxin is defined herein as comprising amino acidresidues 861 through 1296 of SEQ ID NO:28. The receptor-binding domainfor C. botulinum type B toxin is defined herein as comprising amino acidresidues 848 through 1291 of SEQ ID NO:40 (strain Eklund 17B). Thereceptor-binding domain of C. botulinum type C1 toxin is defined hereinas comprising amino acid residues 856 through 1291 of SEQ ID NO:60. Thereceptor-binding domain of C. botulinum type D toxin is defined hereinas comprising amino acid residues 852 through 1276 of SEQ ID NO:66. Thereceptor-binding domain of C. botulinum type E toxin is defined hereinas comprising amino acid residues 835 through 1250 of SEQ ID NO:50(Beluga strain). The receptor-binding domain of C. botulinum type Ftoxin is defined herein as comprising amino acid residues 853 through1274 of SEQ ID NO:71. The receptor-binding domain of C. botulinum type Gtoxin is defined herein as comprising amino acid residues 853 through1297 of SEQ ID NO:77. Within a given serotype, small variations in theprimary amino acid sequence of the botulinal toxins isolated fromdifferent strains has been reported [Whelan et al. (1992), supra andMinton (1995) Curr. Top. Microbiol. Immunol. 195:161-194]. The presentinvention contemplates fusion proteins comprising the receptor-bindingdomain of C. botulinum toxins from serotypes A-G including the variantsfound among different strains within a given serotype. Thereceptor-binding domains listed above are used as the prototype for eachstrain within a serotype. Fusion proteins containing an analogous regionfrom a strain other than the prototype strain are encompassed by thepresent invention.

[0103] Fusion proteins comprising the receptor binding domain (i.e., Cfragment) of botulinal toxins may include amino acid residues locatedbeyond the termini of the domains defined above. For example, thepHisBotB protein contains amino acid residues 846-1291 of SEQ ID NO:40;this fusion protein thus comprises the receptor-binding domain for C.botulinum type B toxin as defined above (i.e., Ile-848 throughGlu-1291). Similarly, pHisBotE contains amino acid residues 827-1252 ofSEQ ID NO:50 and pHisBotG contains amino acid residues 851-1297 of SEQID NO:77. Thus, both pHisBotE and pHisBotG fusion proteins contain a fewamino acids located beyond the N-terminus of the definedreceptor-binding domain.

[0104] The terms “native gene” or “native gene sequences” are used toindicate DNA sequences encoding a particular gene which contain the sameDNA sequences as found in the gene as isolated from nature. In contrast,“synthetic gene sequences” are DNA sequences which are used to replacethe naturally occurring DNA sequences when the naturally occurringsequences cause expression problems in a given host cell. For example,naturally-occurring DNA sequences encoding codons which are rarely usedin a host cell may be replaced (e.g., by site-directed mutagenesis) suchthat the synthetic DNA sequence represents a more frequently used codon.The native DNA sequence and the synthetic DNA sequence will preferablyencode the same amino acid sequence.

SUMMARY OF THE INVENTION

[0105] The present invention relates to the production of polypeptidesderived from toxins particularly in recombinant host cells: In oneembodiment, the present invention provides a host cell containing arecombinant expression vector, said vector encoding a protein comprisingat least a portion of a Clostridium botulinum toxin, said toxin selectedfrom the group consisting of type B toxin and type E toxin. The presentinvention is not limited by the nature of sequences encoding portions ofthe C. botulinum toxin. These sequences may be derived from the nativegene sequences or alternatively they may comprise synthetic genesequences. Synthetic gene sequences are employed when expression of thenative gene sequences is problematic in a given host cell (e.g., whenthe native gene sequences contain sequences resembling yeasttranscription termination signals and the desired host cell is a yeastcell).

[0106] In one embodiment, the host cell is capable of expressing therecombinant C. botulinum toxin protein at a level greater than or equalto 2% to 40% of the total cellular protein and preferably at a levelgreater than or equal to 5% of the total cellular protein. In anotherembodiment, the host cell is capable of expressing the recombinant C.botulinum toxin protein as a soluble protein at a level greater than orequal to 0.25% of the total cellular protein and preferably at a levelgreater than or equal to 0.25% to 10% of the total cellular protein.

[0107] The present invention is not limited by the nature of the hostcell employed for the production of recombinant C. botulinum toxinproteins. In a preferred embodiment, the host cell is an E. coli cell.In another preferred embodiment, the host cell is an insect cell;particularly preferred insect host cells are Spodoptera frugiperda (Sf9)cells. In another preferred embodiment, the host cell is a yeast cell;particularly preferred yeast cells are Pichia pastoris cells.

[0108] In another embodiment, the invention provides a host cellcontaining a recombinant expression vector, said vector encoding afusion protein comprising a non-toxin protein sequence and at least aportion of a Clostridium botulinum toxin, said toxin selected from thegroup consisting of type B toxin and type E toxin. The invention is notlimited by the nature of the portion of the Clostridium botulinum toxinselected. In a preferred embodiment, the portion of the toxin comprisesthe receptor binding domain (i.e., the C fragment of the toxin). Thepresent invention is not limited by the nature of the non-toxin proteinsequence employed. In a preferred embodiment, the non-toxin proteinsequence comprises a poly-histidine tract. A number of alternativefusion tags or fusion partners are known to the art (e.g., MBP, GST,protein A, etc.) and may be employed for the production of fusionproteins comprising a portion of a botulinal toxin.

[0109] The present invention further provides a vaccine comprising afusion protein, said fusion protein comprising a non-toxin proteinsequence and at least a portion of a Clostridium botulinum toxin, saidtoxin selected from the group consisting of type B toxin and type Etoxin. The vaccine may be a monovalent vaccine (i.e., containing only atoxin B fusion protein or a toxin E fusion protein), a bivalent vaccine(i.e., containing both a toxin B fusion protein and a toxin E fusionprotein) or a trivalent or higher valency vaccine. In a preferredembodiment, the toxin B fusion protein and/or toxin E fusion protein iscombined with a fusion protein comprising a non-toxin protein sequenceand at least a portion of Clostridium botulinum type A toxin. Thepresent invention is not limited by the nature of the portion of theClostridium botulinum toxin selected. In a preferred embodiment, theportion of the toxin comprises the receptor binding domain (i.e., the Cfragment of the toxin). The present invention is not limited by thenature of the non-toxin protein sequence employed. In a preferredembodiment, the non-toxin protein sequence comprises a poly-histidinetract. A number of alternative fusion tags or fusion partners are knownto the art (e.g., MBP, GST, protein A, etc.) and may be employed for thegeneration of fusion proteins comprising vaccines. When a fusion partner(i.e., the non-toxin protein sequence) is employed for the production ofa recombinant C. botulinal toxin protein, the fusion partner may beremoved from the recombinant C. botulinal toxin protein if desired(i.e., prior to administration of the protein to a subject) using avariety of methods known to the art (e.g., digestion of fusion proteinscontaining Factor Xa or thrombin recognition sites with the appropriateenzyme). A number of the pETHis vectors employed herein provide anN-terminal his-tag followed by a Factor Xa cleavage site (see Example28a); the botulinal C fragment sequences follow the Factor Xa site andthus, Factor Xa can be used to remove the his-tag from the botulinalfusion protein. In a preferred embodiment, the vaccine is substantiallyendotoxin-free.

[0110] The present invention is not limited by the method employed forthe generation of vaccine comprising fusion proteins comprising anon-toxin protein sequence and at least a portion of a Clostridiumbotulinum toxin. The fusion proteins may be produced by recombinant DNAmeans using either native or synthetic gene sequences expressed in ahost cell. The present invention is not limited to the production ofvaccines using recombinant host cells; cell free in vitrotranscription/translation systems may be employed for the expression ofthe nucleic acid constructs encoding the fusion proteins of the presentinvention. An example of such a cell-free system is the commerciallyavailable TnT™ Coupled Reticulocyte Lysate System (Promega Corporation,Madison, Wis.). Alternatively, the fusion proteins of the presentinvention may be generated by synthetic means (i.e., peptide synthesis).

[0111] The present invention further provides a method of generatingantibody directed against a Clostridium botulinum toxin comprising: a)providing in any order: i) an antigen comprising a fusion proteincomprising a non-toxin protein sequence and at least a portion of aClostridium botulinum toxin, said toxin selected from the groupconsisting of type B toxin and type E toxin, and ii) a host; and b)immunizing the host with the antigen so as to generate an antibody. In apreferred embodiment, the antigen used to immunize the host alsocontains a fusion protein comprising a non-toxin protein sequence and atleast a portion of Clostridium botulinum type A toxin. The presentinvention is not limited by the nature of the portion of the Clostridiumbotulinum toxin selected. In a preferred embodiment, the portion of thetoxin comprises the receptor binding domain (i.e., the C fragment of thetoxin). The present invention is not limited by the nature of thenon-toxin protein sequence employed. In a preferred embodiment, thenon-toxin protein sequence comprises a poly-histidine tract. A number ofalternative fusion tags or fusion partners are known to the art (e.g.,MBP, GST, protein A, etc.) and may be employed for the generation offusion proteins comprising vaccines. When a fusion partner (i.e., thenon-toxin protein sequence) is employed for the production of arecombinant C. botulinal toxin protein, the fusion partner may beremoved from the recombinant C. botulinal toxin protein if desired(i.e., prior to administration of the protein to a subject) using avariety of methods known to the art (e.g., digestion of fusion proteinscontaining Factor Xa or thrombin recognition sites with the appropriateenzyme).

[0112] The present invention is not limited by the nature of the hostemployed for the production of the antibodies of the invention. In apreferred embodiment, the host is a mammal, preferably a human. Theantibodies of the present invention may be generated using non-mammalianhosts such as birds, preferably chickens. In a preferred embodiment themethod of the present invention further comprised the step c) ofcollecting the antibodies from the host. In yet another embodiment, themethod of the present invention further comprises the step d) ofpurifying the antibodies.

[0113] The present invention further provides antibodies raisedaccording to the above methods.

[0114] The present invention further contemplates multivalent vaccinescomprising at least two recombinant C. botulinum toxin proteins derivedfrom the group consisting of C. botulinum serotypes A, B, C, D, E, F,and G. The invention contemplates bivalent, trivalent, quadravalent,pentavalent, heptavalent and septivalent vaccines comprising recombinantC. botulinum toxin proteins. Preferably the recombinant C. botulinumtoxin protein comprises the receptor binding domain (i.e., C fragment)of the toxin.

DESCRIPTION OF THE INVENTION

[0115] The present invention contemplates vaccinating humans and otheranimals with polypeptides derived from C. botulinum neurotoxins whichare substantially endotoxin-free. These botulinal peptides are alsouseful for the production of antitoxin. Anti-botulinal toxin antitoxinis useful for the treatment of patients effected by or at risk ofsymptoms due to the action of C. botulinum toxins. The organisms, toxinsand individual steps of the present invention are described separatelybelow.

[0116] I. Clostridium Species, Clostridial Diseases and AssociatedToxins

[0117] A preferred embodiment of the method of the present invention isdirected toward obtaining antibodies against Clostridium species, theirtoxins, enzymes or other metabolic by-products, cell wall components, orsynthetic or recombinant versions of any of these compounds. It iscontemplated that these antibodies will be produced by immunization ofhumans or other animals. It is not intended that the present inventionbe limited to any particular toxin or any species of organism. In oneembodiment, toxins from all Clostridium species are contemplated asimmunogens. Examples of these toxins include the neuraminidase toxin ofC. butyricum, C. sordellii toxins HT and LT, toxins A, B, C, D, E; F,and G of C. botulinum and the numerous C. perfringens toxins. In onepreferred embodiment, toxins A, B, and E of C. botulinum arecontemplated as immunogens. Table 2 above lists various Clostridiumspecies, their toxins and some antigens associated with disease. TABLE 2Clostridial Toxins Organism Toxins and Disease-Associated Antigens C.botulinum A, B, C₁, C₂, D, E, F, G C. butyricum Neuraminidase C.difficile A, B, Enterotoxin (not A nor B), Motility Altering Factor, LowMolecular Weight Toxin, Others C. perfringens α, β, ε, ι, γ, δ, ν, θ, κ,λ, μ, υ C. sordelli/ HT, LT, α, β, γ C. bifermentans C. novyi α, β, γ,δ, ε, ζ, ν, θ C. septicum α, β, γ, δ C. histolyticum α, β, γ, δ, ε plusadditional enzymes C. chauvoei α, β, γ, δ

[0118] It is not intended that antibodies produced against one toxinwill only be used against that toxin. It is contemplated that antibodiesdirected against one toxin (e.g. C. perfringens type A enterotoxin) maybe used as an effective therapeutic against one or more toxin(s)produced by other members of the genus Clostridium or other toxinproducing organisms (e.g., Bacillus cereus, Staphylococcus aureus,Streptococcus mutans, Acinetobacter calcoaceticus, Pseudomonasaeruginosa, other Pseudomonas species, etc.). It is further contemplatedthat antibodies directed against the portion of the toxin which binds tomammalian membranes (e.g., C. perfringens enterotoxin A) can also beused against other organisms. It is contemplated that these membranebinding domains are produced synthetically and used as immunogens.

[0119] II. Obtaining Antibodies in Non-Mammals

[0120] A preferred embodiment of the method of the present invention forobtaining antibodies involves immunization. However, it is alsocontemplated that antibodies could be obtained from non-mammals withoutimmunization. In the case where no immunization is contemplated, thepresent invention may use non-mammals with preexisting antibodies totoxins as well as non-mammals that have antibodies to whole organisms byvirtue of reactions with the administered antigen. An example of thelatter involves immunization with synthetic peptides or recombinantproteins sharing epitopes with whole organism components.

[0121] In a preferred embodiment, the method of the present inventioncontemplates immunizing non-mammals with bacterial toxin(s). It is notintended that the present invention be limited to any particular toxin.In one embodiment, toxin from all clostridial bacteria sources (seeTable 2) are contemplated as immunogens. Examples of these toxins are C.butyricum neuraminidase toxin, toxins A, B, C, D, E, F, and G from C.botulinum, C. perfringens toxins α, β, ε, and ι and C. sordelii toxinsHT and LT. In a preferred embodiment, C. botulinum toxins A, B, C, D. E,and F (or fragments thereof) are contemplated as immunogens.

[0122] A particularly preferred embodiment involves the use of bacterialtoxin protein or fragments of toxin proteins produced by molecularbiological means (i.e., recombinant toxin proteins). In a preferredembodiment, the immunogen comprises the receptor-binding domain (i.e.,the ˜50 kD carboxy-terminal portion of the heavy chain; also referred toas the C fragment) of C. botulinum serotype A neurotoxin produced byrecombinant DNA technology. In another preferred embodiment, theimmunogen comprises the receptor-binding domain of C. botulinum serotypeB neurotoxin produced by recombinant DNA technology. In yet anotherpreferred embodiment, the immunogen comprises the receptor-bindingdomain region of C. botulinum serotype E neurotoxin produced byrecombinant DNA technology. In yet another preferred embodiment, theimmunogen comprises the receptor-binding domain region of C. botulinumserotype C1 neurotoxin produced by recombinant DNA technology. In yetanother preferred embodiment, the immunogen comprises thereceptor-binding domain region of C. botulinum serotype C2 neurotoxinproduced by recombinant DNA technology. In yet another preferredembodiment, the immunogen comprises the receptor-binding domain regionof C. botulinum serotype D neurotoxin produced by recombinant DNAtechnology. In yet another preferred embodiment, the immunogen comprisesthe receptor-binding domain region of C. botulinum serotype F neurotoxinproduced by recombinant DNA technology. In yet another preferredembodiment, the immunogen comprises the receptor-binding domain regionof C. botulinum serotype G neurotoxin produced by recombinant DNAtechnology. In a preferred embodiment, the recombinant botulinal toxinproteins are expressed as fusion proteins (e.g., as histidine-taggedproteins). In a still further preferred embodiment, the immunogen is amultivalent vaccine comprising the receptor-binding domain region of C.botulinum toxin from two or more toxins selected from the groupconsisting of type A, type B, type C (including C1 and C2), type D, typeE, and type F toxin.

[0123] When immunization is used, the preferred non-mammal is from theclass Aves. All birds are contemplated (e.g., duck, ostrich, emu,turkey, etc.). A preferred bird is a chicken. Importantly, chickenantibody does not fix mammalian complement. [See H. N. Benson et al., J.Immunol. 87:616 (1961).] Thus, chicken antibody will normally not causea complement-dependent reaction. [A. A. Benedict and K. Yamaga,“Immunoglobulins and Antibody Production in Avian Species,” inComparative Immunology (J. J. Marchaloni, ed.), pp. 335-375, Blackwell,Oxford (1966).] Thus, the preferred antitoxins of the present inventionwill not exhibit complement-related side effects observed withantitoxins known presently.

[0124] When birds are used, it is contemplated that the antibody will beobtained from either the bird serum or the egg. A preferred embodimentinvolves collection of the antibody from the egg. Laying hens transportimmunoglobulin to the egg yolk (“IgY”) in concentrations equal to orexceeding that found in serum. [See R. Patterson et al., J. Immunol.89:272 (1962); and S. B. Carroll and B. D. Stollar, J. Biol. Chem.258:24 (1983).] In addition, the large volume of egg yolk producedvastly exceeds the volume of serum that can be safely obtained from thebird over any given time period. Finally, the antibody from eggs ispurer and more homogeneous; there is far less non-immunoglobulin protein(as compared to serum) and only one class of immunoglobulin istransported to the yolk.

[0125] When considering immunization with toxins, one may considermodification of the toxins to reduce the toxicity. In this regard, it isnot intended that the present invention be limited by immunization withmodified toxin. Unmodified (“native”) toxin is also contemplated as animmunogen.

[0126] It is also not intended that the present invention be limited bythe type of modification—if modification is used. The present inventioncontemplates all types of toxin modification, including chemical andheat treatment of the toxin. The preferred modification, however, isformaldehyde treatment.

[0127] It is not intended that the present invention be limited to aparticular mode of immunization; the present invention contemplates allmodes of immunization, including subcutaneous, intramuscular,intraperitoneal, and intravenous or intravascular injection, as well asper os administration of immunogen.

[0128] The present invention further contemplates immunization with orwithout adjuvant. (Adjuvant is defined as a substance known to increasethe immune response to other antigens when administered with otherantigens.) If adjuvant is used, it is not intended that the presentinvention be limited to any particular type of adjuvant—or that the sameadjuvant, once used, be used all the time. While the present inventioncontemplates all types of adjuvant, whether used separately or incombinations, the preferred use of adjuvant is the use of CompleteFreund's Adjuvant followed sometime later with Incomplete Freund'sAdjuvant. Another preferred use of adjuvant is the use of GerbuAdjuvant. The invention also contemplates the use of RIBI fowl adjuvantand Quil A adjuvant.

[0129] When immunization is used, the present invention contemplates awide variety of immunization schedules. In one embodiment, a chicken isadministered toxin(s) on day zero and subsequently receives toxin(s) inintervals thereafter. It is not intended that the present invention belimited by the particular intervals or doses. Similarly, it is notintended that the present invention be limited to any particularschedule for collecting antibody. The preferred collection time issometime after day 100.

[0130] Where birds are used and collection of antibody is performed bycollecting eggs, the eggs may be stored prior to processing forantibody. It is preferred that eggs be stored at 4° C. for less than oneyear.

[0131] It is contemplated that chicken antibody produced in this mannercan be buffer-extracted and used analytically. While unpurified, thispreparation can serve as a reference for activity of the antibody priorto further manipulations (e.g., immunoaffinity purification).

[0132] III. Increasing the Effectiveness of Antibodies

[0133] When purification is used, the present invention contemplatespurifying to increase the effectiveness of both non-mammalian antitoxinsand mammalian antitoxins. Specifically, the present inventioncontemplates increasing the percent of toxin-reactive immunoglobulin.The preferred purification approach for avian antibody is polyethyleneglycol (PEG) separation.

[0134] The present invention contemplates that avian antibody beinitially purified using simple, inexpensive procedures. In oneembodiment, chicken antibody from eggs is purified by extraction andprecipitation with PEG. PEG purification exploits the differentialsolubility of lipids (which are abundant in egg yolks) and yolk proteinsin high concentrations of PEG 8000. [Polson et al., Immunol. Comm. 9:495(1980).] The technique is rapid, simple, and relatively inexpensive andyields an immunoglobulin fraction that is significantly purer in termsof contaminating non-immunoglobulin proteins than the comparableammonium sulfate fractions of mammalian sera and horse antibodies. Themajority of the PEG is removed from the precipitated chickenimmunoglobulin by treatment with ethanol. Indeed, PEG-purified antibodyis sufficiently pure that the present invention contemplates the use ofPEG-purified antitoxins in the passive immunization of intoxicatedhumans and animals.

[0135] IV. Treatment

[0136] The present invention contemplates antitoxin therapy for humansand other animals intoxicated by bacterial toxins. A preferred method oftreatment is by intravenous administration of anti-boutlinal antitoxin;oral administration is also contemplated for other clostridialantitoxins.

[0137] A. Dosage of Antitoxin

[0138] It was noted by way of background that a balance must be struckwhen administering currently available antitoxin which is usuallyproduced in large animals such as horses; sufficient antitoxin must beadministered to neutralize the toxin, but not so much antitoxin as toincrease the risk of untoward side effects. These side effects arecaused by: i) patient sensitivity to foreign (e.g, horse) proteins; ii)anaphylactic or immunogenic properties of non-immunoglobulin proteins;iii) the complement fixing properties of mammalian antibodies; and/oriv) the overall burden of foreign protein administered. It is extremelydifficult to strike this balance when, as noted above, the degree ofintoxication (and hence the level of antitoxin therapy needed) can onlybe approximated.

[0139] The present invention contemplates significantly reducing sideeffects so that this balance is more easily achieved. Treatmentaccording to the present invention contemplates reducing side effects byusing PEG-purified antitoxin from birds.

[0140] In one embodiment, the treatment of the present inventioncontemplates the use of PEG-purified antitoxin from birds. The use ofyolk-derived, PEG-purified antibody as antitoxin allows for theadministration of: 1) non(mammalian)-complement-fixing, avian antibody;2) a less heterogeneous mixture of non-immunoglobulin proteins; and 3)less total protein to deliver the equivalent weight of active antibodypresent in currently available antitoxins. The non-mammalian Source ofthe antitoxin makes it useful for treating patients who are sensitive tohorse or other mammalian sera.

[0141] B. Delivery of Antitoxin

[0142] Although it is not intended to limit the route of delivery, thepresent invention contemplates a method for antitoxin treatment ofbacterial intoxication in which delivery of antitoxin is oral. In oneembodiment, antitoxin is delivered in a solid form (e.g., tablets). Inan alternative embodiment antitoxin is delivered in an aqueous solution.When an aqueous solution is used, the solution has sufficient ionicstrength to solubilize antibody protein, yet is made palatable for oraladministration. The delivery solution may also be buffered (e.g.,carbonate buffer pH 9.5) which can neutralize stomach acids andstabilize the antibodies when the antibodies are administered orally. Inone embodiment the delivery solution is an aqueous solution. In anotherembodiment the delivery solution is a nutritional formula. Preferably,the delivery solution is infant formula. Yet another embodimentcontemplates the delivery of lyophilized antibody encapsulated ormicroencapsulated inside acid-resistant compounds.

[0143] Methods of applying enteric coatings to pharmaceutical compoundsare well known to the art [companies specializing in the coating ofpharmaceutical compounds are available; for example, The Coating Place(Verona, Wis.) and AAI (Wilmington, N.C.)]. Enteric coatings which areresistant to gastric fluid and whose release (i.e., dissolution of thecoating to release the pharmaceutical compound) is pH dependent arecommercially available [for example, the polymethacrylates Eudragit® Land Eudragit® S(Röhm GmbH)]. Eudragit® S is soluble in intestinal fluidfrom pH 7.0; this coating can be used to microencapsulate lyophilizedantitoxin antibodies and the particles are suspended in a solutionhaving a pH above or below pH 7.0 for oral administration. Themicroparticles will remain intact and undissolved until they reached theintestines where the intestinal pH would cause them to dissolve therebyreleasing the antitoxin.

[0144] The invention contemplates a method of treatment which can beadministered for treatment of acute intoxication. In one embodiment,antitoxin is administered orally in either a delivery solution or intablet form, in therapeutic dosage, to a subject intoxicated by thebacterial toxin which served as immunogen for the antitoxin.

[0145] The invention also contemplates a method of treatment which canbe administered prophylactically. In one embodiment, antitoxin isadministered orally, in a delivery solution, in therapeutic dosage, to asubject, to prevent intoxication of the subject by the bacterial toxinwhich served as immunogen for the production of antitoxin. In anotherembodiment, antitoxin is administered orally in solid form such astablets or as microencapsulated particles. Microencapsulation oflyophilized antibody using compounds such as Eudragit® (Rohm GmbH) orpolyethylene glycol, which dissolve at a wide range of pH units, allowsthe oral administration of solid antitoxin in a liquid form (i.e., asuspension) to recipients unable to tolerate administration of tablets(e.g., children or patients on feeding tubes). In one preferredembodiment the subject is a child. In another embodiment, antibodyraised against whole bacterial organism is administered orally to asubject, in a delivery solution, in therapeutic dosage.

[0146] V. Vaccines Against Clostridial Species

[0147] The invention contemplates the generation of mono- andmultivalent vaccines for the protection of an animal (particularlyhumans) against several clostridial species. Of particular interest arevaccines which stimulate the production of a humoral immune response toC. botulinum, C. tetani and C. difficile in humans. The antigenscomprising the vaccine preparation may be native or recombinantlyproduced toxin proteins from the clostridial species listed above. Whentoxin proteins are used as immunogens they are generally modified toreduce the toxicity. This modification may be by chemical or genetic(i.e., recombinant DNA technology) means. In general geneticdetoxification (i.e., the expression of nontoxic fragments in a hostcell) is preferred as the expression of nontoxic fragments in a hostcell precludes the presence of intact, active toxin in the finalpreparation. However, when chemical modification is desired, thepreferred toxin modification is formaldehyde treatment.

[0148] The invention contemplates that recombinant C. botulinum toxinproteins be used as antigens in mono- and multivalent vaccinepreparations. Soluble, substantially endotoxin-free recombinant C.botulinum toxin proteins derived from serotypes A, B and E may be usedindividually (i.e., as mono-valent vaccines) or in combination (i.e., asa multi-valent vaccine). In addition, the recombinant C. botulinum toxinproteins derived from serotpes A, B and E may be used in conjunctionwith either recombinant or native toxins or toxoids from other serotypesof C. botulinum, C. difficile and C. tetani as antigens for thepreparation of these mono- and multivalent vaccines. It is contemplatedthat, due to the structural similarity of C. botulinum and C. tetanitoxin proteins, a vaccine comprising C. difficile and botulinum toxinproteins (native or recombinant or a mixture thereof be used tostimulate an immune response against C. botulinum, C. tetani and C.difficile.

[0149] The present invention further contemplates multi-valent vaccinescomprising two or more botulinal toxin proteins selected from the groupcomprising recombinant C. botulinum toxin proteins derived fromserotypes A, B, C (including C1 and C2), D, E, F and G.

[0150] The adverse consequences of exposure to botulinal toxin would beavoided by immunization of subjects at risk of exposure to the toxinwith nontoxic preparations which confer immunity such as chemically orgenetically detoxified toxin.

[0151] Vaccines which confer immunity against one or more of the toxintypes A, B, E, F and G would be useful as a means of protecting humansfrom the deleterious effects of those C. botulinum toxins known toaffect man. Indeed as the possibility exists that humans could beexposed to any of the seven serotypes of C. botulinum toxin (e.g.,during biological warfare or the production of toxin in a laboratorysetting), multivalent vaccines capable of conferring immunity againsttoxin types A-G (including both C1 and C2 toxins) would be useful forthe protection of humans. Vaccines which confer immunity against one ormore of the toxin types C, D and E would be useful for veterinaryapplications.

[0152] The botulinal neurotoxin is synthesized as a single polypeptidechain which is processed into a heavy (H; ˜100 kD) and a light (L; ˜50kD) chain by cleavage with proteolytic enzymes; these two chains areheld together via disulfide bonds in the active toxin (referred to asderivative toxin) [B. R. DasGupta and H. Sugiyama, Biochem. Biophys.Res. Commun. 48:108 (1972); reviewed in B. R. DasGupta, J. Physiol.84:220 (1990), H. Sugiyama, Microbiol. Rev. 44:419 (1980) and C. L.Hatheway, Clin. Microbiol. Rev. 3:66 (1990)]. The heavy chain of theactive toxin is cleaved by trypsin to produce two fragments termed H_(C)(also referred to as H₁ or C) and H_(N) (also referred to as H₂ or B).The H_(C) fragment (˜46 kD) comprises the carboxy end of the H chain.The H_(N) fragment (˜49 kD) comprises the animo end and remains attachedto the L chain (H_(N)L). Neither H_(C) or H_(N)L is toxic. H_(C)competes with whole derivative toxin for binding to synaptosomes andtherefore H_(C) is said to contain the receptor binding site. The H_(C)and H_(N) fragments of botulinal toxin are analogous to the fragments Cand B of tetanus toxin which are produced by papain cleavage. The Cfragment of tetanus toxin has been shown to be responsible for thebinding of tetanus toxin to purified gangliosides and neuronal cells[Halpern and Loftus, J. Biol. Chem. 288:11188 (1993)].

[0153] Antisera raised against purified preparations of isolatedbotulinal H and L chains have been shown to protect mice against thelethal effects of the toxin; however, the effectiveness of the twoantisera differ with the anti-H sera being more potent (H. Sugiyama,supra). While the different botulinal toxins show structural similarityto one another, the different serotypes are reported to beimmunologically distinct (i.e., sera raised against one toxin type doesnot cross-react to a significant degree with other types). Thus, thegeneration of multivalent vaccines may require the use of more than onetype of toxin.

[0154]C. botulinum toxin genes from all seven serotypes have been clonedand sequenced (Minton (1995), supra); in addition, partial amino acidsequence is available for a number of C. botulinum toxins isolated fromdifferent strains within a given serotype. The C. botulinum toxinscontain about 1250-1300 amino acid residues. On the DNA level, theoverall degree of homology between C. botulinum serotypes A, B, C, D andE toxins averages between 50 and 60% identity with a greater degree ofhomology being found between H chain-encoding regions than between thoseencoding L chains [Whelan et al. (1992) Appl. Environ. Microbiol.58:2345]. The degree of identity between C. botulinum toxins on theamino acid level reflects the level of DNA sequence homology. The mostdivergent area of DNA and amino acid sequence is found within thecarboxy-terminal area of the various C. botulinum H chain genes. Thisportion of the toxin (i.e., H_(C) or the C fragment) plays a major rolein cell binding. As toxin from different serotypes is thought to bind todistinct cell receptor molecules, it is not surprising that the toxinsdiverge significantly over this region.

[0155] Within a given serotype, small variations in the primary aminoacid sequence of the botulinal toxins isolated from different strainshas been reported [Whelan et al. (1992), supra and Minton (1995),supra]. The present invention contemplates fusion proteins comprisingportions of C. botulinum toxins from serotypes A-G including thevariants found among different strains within a given serotype. Thepresent invention provides oligonucleotide primers which may be used toamplify the C fragment or receptor-binding region of the toxin gene fromvarious strains of C. botulinum serotype A, serotype B, serotype C(C1and C2), serotype D, serotype E, serotype F and serotype G. A largenumber of different strains of C. botulinum serotype A, serotype B,serotype C, serotype D serotype E and serotype F are available from theAmerican Type Culture Collection (ATCC; Rockville, Md.). For example,the ATCC provides the following: Type A strains: 174 (ATCC 3502), 457(ATCC 17862), and NCTC 7272 (ATCC 19397); Type B strains: 34 (ATCC 439),62A (ATCC 7948), NCA 213 B (ATCC 7949), 13114 (ATCC 8083), 3137 (ATCC17780), 1347 (ATCC 17841), 2017 (ATCC 17843), 2217 (ATCC 17844), 2254(ATCC 17845) and VP 1731 (ATCC 25765); Type C strains: 2220 (ATCC17782), 2239 (ATCC 17783), 2223 (ATCC 17784; a type C-β strain; C-βstrains produce C2 toxin), 662 (ATCC 17849; a type C-α strain; C-αstrains produce mainly C1 toxin and a small amount of C2 toxin), 2021(ATCC 17850; a type C-α strain) and VPI 3803 (ATCC 25766); Type Dstrains: ATCC 9633, 2023 (ATCC 17851), and VPI 5995 (ATCC 27517); Type Estrains: ATCC 43181, 36208 (ATCC 9564), 2231 (ATCC 17786), 2229 (ATCC17852), 2279 (ATCC 17854) and 2285 (ATCC 17855) and Type F strains: 202F(ATCC 23387), VPI 4404 (ATCC 25764), VPI 2382 (ATCC 27321) and Langeland(ATCC 35415). Type G strain, 113/30 (NCFB 3012) may be obtained from theNational Collection of Food Bacteria (NCFB, AFRC Institute of FoodResearch, Reading, United Kingdom).

[0156] Purification methods have been reported for native toxin types A,B, C, D, E, and F [reviewed in G. Sakaguchi, Pharmac. Ther. 19:165(1983)]. As the different botulinal toxins are structurally related, theinvention contemplates the expression of any of the botulinal toxins(e.g., types A-G) as soluble recombinant fusion proteins.

[0157] In particular, methods for purification of the type A botulinumneurotoxin have been developed [L. J. Moberg and H. Sugiyama, Appl.Environ. Microbiol. 35:878 (1978)]. Immunization of hens with detoxifiedpurified protein results in the generation of neutralizing antibodies[B. S. Thalley et al., in Botulinum and Tetanus Neurotoxins, B. R.DasGupta, ed., Plenum Press, New York (1993), p. 467].

[0158] The currently available C. botulinum pentavalent vaccinecomprising chemically inactivated (i.e., formaldehyde treated) type A,B, C, D and E toxins is not adequate. The efficacy is variable (inparticular, only 78% of recipients produce protective levels ofanti-type B antibodies following administration of the primary series)and immunization is painful (deep subcutaneous inoculation is requiredfor administration), with adverse reactions being common (moderate tosevere local reactions occur in approximately 6% of recipients uponinitial injection; this number rises to approximately 11% of individualswho receive booster injections) [Informational Brochure for thePentavalent (ABCDE) Botulinum Toxoid, Centers for Disease Control].Preparation of this vaccine is dangerous as active toxin must be handledby laboratory workers.

[0159] In general, chemical detoxification of bacterial toxins usingagents such as formaldehyde, glutaraldehyde or hydrogen peroxide is notoptimal for the generation of vaccines or antitoxins. A delicate balancemust be struck between too much and too little chemical modification. Ifthe treatment is insufficient, the vaccine may retain residual toxicity.If the treatment is too excessive, the vaccine may lose potency due todestruction of native immunogenic determinants. Another major limitationof using botulinal toxoids for the generation of antitoxins or vaccinesis the high production expense. For the above reasons, the developmentof methods for the production of nontoxic but immunogenic C. botulinumtoxin proteins is desirable.

[0160] The C. botulinum and C. tetanus toxin proteins have similarstructures [reviewed in E. J. Schantz and E. A. Johnson, Microbiol. Rev.56:80 (1992)]. The carboxy-terminal 50 kD fragment of the tetanus toxinheavy chain (fragment C) is released by papain cleavage and has beenshown to be non-toxic and immunogenic. Recombinant tetanus toxinfragment C has been developed as a candidate vaccine antigen [A. J.Makoff et al., Bio/Technology 7:1043 (1989)]. Mice immunized withrecombinant tetanus toxin fragment C were protected from challenge withlethal doses of tetanus toxin. No studies have demonstrated that therecombinant tetanus fragment C protein confers immunity against otherbotulinal toxins such as the C. botulinum toxins.

[0161] Recombinant tetanus fragment C has been expressed in E. coli (A.J. Makoff et al., Bio/Technology, supra and Nucleic Acids Res. 17:10191(1989); J. L. Halpern et al., Infect. Immun. 58:1004 (1990)], yeast [M.A. Romanos et al., Nucleic Acids Res. 19:1461 (1991)] and baculovirus[I. G. Charles et al., Infect. Immun. 59:1627 (1991)]. Synthetic tetanustoxin genes had to be constructed to facilitate expression in yeast (M.A. Romanos et al., supra) and E. coli [A. J. Makoff et al., NucleicAcids Res., supra], due to the high A-T content of the tetanus toxingene sequences. High A-T content is a common feature of clostridialgenes (M. R. Popoff et al., Infect. Immun. 59:3673 (1991); H. F.LaPenotiere et al., in Botulinum and Tetanus Neurotoxins, B. R.DasGupta, ed., Plenum Press, New York (1993), p. 463] which createsexpression difficulties in E. coli and yeast due primarily to alteredcodon usage frequency and fortuitous polyadenylation sites,respectively.

[0162] The C fragment of the C. botulinum type A neurotoxin heavy chainhas been evaluated as a vaccine candidate. The C. botulinum type Aneurotoxin gene has been cloned and sequenced [D. E. Thompson et al.,Eur. J. Biochem. 189:73 (1990)]. The C fragment of the type A toxin wasexpressed as either a fusion protein comprising the botulinal C fragmentfused with the maltose binding protein (MBP) or as a native protein [H.F. LaPenotiere et al., (1993) supra, H. F. LaPenotiere et al., Toxicon.33:1383 (1995) and Middlebrook and Brown (1995), Curr. Top. Microbiol.Immunol. 195:89-122]. The plasmid construct encoding the native proteinwas reported to be unstable, while the fusion protein was expressedprimarily in inclusion bodies as insoluble protein. Immunization of micewith crudely purified MBP fusion protein resulted in protection againstIP challenge with 3 LD₅₀ doses of toxin [LaPenotiere et al., (1993) and(1995), supra]. However, this recombinant C. botulinum type A toxin Cfragment/MBP fusion protein is not a suitable immunogen for theproduction of vaccines as it is expressed as an insoluble protein in E.coli. Furthermore, this recombinant C. botulinum type A toxin Cfragment/MBP fusion protein was not shown to be substantially free ofendotoxin contamination. Experience with recombinant C. botulinum type Atoxin C fragment/MBP fusion proteins shows that the presence of the MBPon the fusion protein greatly complicates the removal of endotoxin frompreparations of the recombinant fusion protein (see Ex. 24, infra).Expression of a synthetic gene encoding C. botulinum type A toxin Cfragment as a soluble protein excreted from insect cells has beenreported [Middlebrook and Brown (1995), supra]; no details regarding thelevel of expression achieved or the presence of endotoxin or otherpyrogens were provided. Like the insoluble protein expressed in E. coli,immunization with the recombinant protein produced in insect cells wasreported to protect mice from challenge with C. botulinum toxin A.

[0163] Inclusion body protein must be solubilized prior to purificationand/or administration to a host. The harsh treatment of inclusion bodyprotein needed to accomplish this solubilization may reduce theimmunogenicity of the purified protein. Ideally, recombinant proteins tobe used as vaccines are expressed as soluble proteins at high levels(i.e., greater than or equal to about 0.75% of total cellular protein)in E. coli or other host cells (e.g., yeast, insect cells, etc.). Thisfacilitates the production and isolation of sufficient quantities of theimmunogen in a highly purified form (i.e., substantially free ofendotoxin or other pyrogen contamination). The ability to expressrecombinant toxin proteins as soluble proteins in E. coli isadvantageous due to the low cost of growth compared to insect ormammalian tissue culture cells.

[0164] The C. botulinum type B neurotoxin gene has been cloned andsequenced from two strains of C. botulinum type B [Whelan et al. (1992)Appl. Environ. Microbiol. 58:2345 (Danish strain) and Hutson et al.(1994) Curr. Microbiol. 28:101 (Eklund 17B strain)]. The nucleotidesequence of the toxin gene derived from the Eklund 17B strain (ATCC25765) is available from the EMBL/GenBank sequence data banks under theaccession number X71343; the nucleotide sequence of the coding region islisted in SEQ ID NO:39. The amino acid sequence of the C. botulinum typeB neurotoxin derived from the strain Eklund 17B is listed in SEQ IDNO:40. The nucleotide sequence of the C. botulinum serotype B toxin genederived from the Danish strain is listed in SEQ ID NO:41. The amino acidsequence of the C. botulinum type B neurotoxin derived from the Danishstrain is listed in SEQ ID NO:42.

[0165] The C. botulinum type B neurotoxin gene is synthesized as asingle polypeptide chain which is processed to form a dimer composed ofa light and a heavy chain linked via disulfide bonds. The light chain isresponsible for pharmacological activity (i.e., inhibition of therelease of acetylcholine at the neuromuscular junction). The N-terminalportion of the heavy chain is thought to mediate channel formation whilethe C-terminal portion mediates toxin binding; the type B neurotoxin hasbeen reported to exist as a mixture of predominantly single chain withsome double chain (Whelan et al., supra). The 50 kD carboxy-terminalportion of the heavy chain is referred to as the C fragment or the H_(C)domain. The present invention reports for the first time, the expressionof the C fragment of C. botulinum type B toxin in heterologous hosts(e.g., E. coli).

[0166] The C. botulinum type E neurotoxin gene has been cloned andsequenced from a number of different strains [Poulet et al. (1992)Biochem. Biophys. Res. Commun. 183:107; Whelan et al. (1992) Eur. J.Biochem. 204:657; and Fujii et al. (1993) J. Gen. Microbiol. 139:79].The nucleotide sequence of the type E toxin gene is available from theEMBL sequence data bank under accession numbers X62089 (strain Beluga)and X62683 (strain NCTC 11219); the nucleotide sequence of the codingregion (strain Beluga) is listed in SEQ ID NO:45. The amino acidsequence of the C. botulinum type E neurotoxin derived from strainBeluga is listed in SEQ ID NO:46. The type E neurotoxin gene issynthesized as a single polypeptide chain which may be converted to adouble-chain form (i.e., a heavy chain and a light chain) by cleavagewith trypsin; unlike the type A neurotoxin, the type E neurotoxin existsessentially only in the single-chain form. The 50 kD carboxy-terminalportion of the heavy chain is referred to as the C fragment or the H_(C)domain. The present invention reports for the first time, the expressionof the C fragment of C. botulinum type E toxin in heterologous hosts(e.g., E. coli).

[0167] The C. botulinum type C1, D, F and G neurotoxin genes have beencloned and sequenced. The nucleotide and amino acid sequences of thesegenes and toxins are provided herein. The invention provides methods forthe expression of the C fragment from each of these toxin genes inheterologous hosts and the purification of the resulting recombinantproteins.

[0168] The subject invention provides methods which allow the productionof soluble C. botulinum toxin proteins in economical host cells (e.g.,E. coli). In addition the subject invention provides methods which allowthe production of soluble botulinal toxin proteins in yeast and insectcells. Further, methods for the isolation of purified soluble C.botulinum toxin proteins which are suitable for immunization of humansand other animals are provided. These soluble, purified preparations ofC. botulinum toxin proteins provide the basis for improved vaccinepreparations and facilitate the production of antitoxin.

[0169] When recombinant clostridial toxin proteins produced ingram-negative bacteria (e.g., E. coli) are used as vaccines, they arepurified to remove endotoxin prior to administration to a host animal.In order to vaccinate a host, an immunogenically-effective amount ofpurified substantially endotoxin-free recombinant clostridial toxinprotein is administered in any of a number of physiologically acceptablecarriers known to the art. When administered for the purpose ofvaccination, the purified substantially endotoxin-free recombinantclostridial toxin protein may be used alone or in conjunction with knownadjutants, including potassium alum, aluminum phosphate, aluminumhydroxide, Gerbu adjuvant (GmDP; C. C. Biotech Corp.), RIBI adjuvant(MPL; RIBI Immunochemical Research, Inc.), QS21 (Cambridge Biotech). Thealum and aluminum-based adjutants are particularly preferred whenvaccines are to be administered to humans; however, any adjuvantapproved for use in humans may be employed. The route of immunizationmay be nasal, oral, intramuscular, intraperitoneal or subcutaneous.

[0170] The invention contemplates the use of soluble, substantiallyendotoxin-free preparations of fusion proteins comprising the C fragmentof the C. botulinum type A, B, C, D, E, F, and G toxin as vaccines. Inone embodiment, the vaccine comprises the C fragment of either the C.botulinum type A, B, C, D, E, F, or G toxin and a poly-histidine tract(also called a histidine tag). In a particularly preferred embodiment, afusion protein comprising the histidine tagged C fragment is expressedusing the pET series of expression vectors (Novagen). The pET expressionsystem utilizes a vector containing the T7 promoter which encodes thefusion protein and a host cell which can be induced to express the T7DNA polymerase (i.e., a DE3 host strain). The production of C fragmentfusion proteins containing a histidine tract is not limited to the useof a particular expression vector and host strain. Several commerciallyavailable expression vectors and host strains can be used to express theC fragment protein sequences as a fusion protein containing a histidinetract (For example, the pQE series (pQE-8, 12, 16, 17, 18, 30, 31, 32,40, 41, 42, 50, 51, 52, 60 and 70) of expression vectors (Qiagen) whichare used with the host strains M15[pREP4] (Qiagen) and SG13009[pREP4](Qiagen) can be used to express fusion proteins containing six histidineresidues at the amino-terminus of the fusion protein). Furthermore anumber of commercially available expression vectors which provide ahistidine tract also provide a protease cleavage site between thehistidine tract and the protein of interest (e.g., botulinal toxinsequences). Cleavage of the resulting fusion protein with theappropriate protease will remove the histidine tag from the protein ofinterest (e.g., botulinal toxin sequences) (see Example 28a, infra).Removal of the histidine tag may be desirable prior to administration ofthe recombinant botulinal toxin protein to a subject (e.g., a human).

[0171] VI. Detection of Toxin

[0172] The invention contemplates detecting bacterial toxin in a sample.The term “sample” in the present specification and claims is used in itsbroadest sense. On the one hand it is meant to include a specimen orculture. On the other hand, it is meant to include both biological andenvironmental samples.

[0173] Biological samples may be animal, including human, fluid, solid(e.g., stool) or tissue; liquid and solid food products and ingredientssuch as dairy items, vegetables, meat and meat by-products, and waste.Environmental samples include environmental material such as surfacematter, soil, water and industrial samples, as well as samples obtainedfrom food and dairy processing instruments, apparatus, equipment,disposable and non-disposable items. These examples are not to beconstrued as limiting the sample types applicable to the presentinvention.

[0174] The invention contemplates detecting bacterial toxin by acompetitive immunoassay method that utilizes recombinant toxin A andtoxin B proteins, antibodies raised against recombinant bacterial toxinproteins. A fixed amount of the recombinant toxin proteins areimmobilized to a solid support (e.g., a microtiter plate) followed bythe addition of a biological sample suspected of containing a bacterialtoxin. The biological sample is first mixed with affinity-purified orPEG fractionated antibodies directed against the recombinant toxinprotein. A reporter reagent is then added which is capable of detectingthe presence of antibody bound to the immobilized toxin protein. Thereporter substance may comprise an antibody with binding specificity forthe antitoxin attached to a molecule which is used to identify thepresence of the reporter substance. If toxin is present in the sample,this toxin will compete with the immobilized recombinant toxin proteinfor binding to the anti-recombinant antibody thereby reducing the signalobtained following the addition of the reporter reagent. A control isemployed where the antibody is not mixed with the sample. This gives thehighest (or reference) signal.

[0175] The invention also contemplates detecting bacterial toxin by a“sandwich” immunoassay method that utilizes antibodies directed againstrecombinant bacterial toxin proteins. Affinity-purified antibodiesdirected against recombinant bacterial toxin proteins are immobilized toa solid support (e.g., microtiter plates). Biological samples suspectedof containing bacterial toxins are then added followed by a washing stepto remove substantially all unbound antitoxin. The biological sample isnext exposed to the reporter substance, which binds to antitoxin and isthen washed free of substantially all unbound reporter substance. Thereporter substance may comprise an antibody with binding specificity forthe antitoxin attached to a molecule which is used to identify thepresence of the reporter substance. Identification of the reportersubstance in the biological tissue indicates the presence of thebacterial toxin.

[0176] It is also contemplated that bacterial toxin be detected bypouring liquids (e.g., soups and other fluid foods and feeds includingnutritional supplements for humans and other animals) over immobilizedantibody which is directed against the bacterial toxin. It iscontemplated that the immobilized antibody will be present in or on suchsupports as cartridges, columns, beads, or any other solid supportmedium. In one embodiment, following the exposure of the liquid to theimmobilized antibody, unbound toxin is substantially removed by washing.The exposure of the liquid is then exposed to a reporter substance whichdetects the presence of bound toxin. In a preferred embodiment thereporter substance is an enzyme, fluorescent dye, or radioactivecompound attached to an antibody which is directed against the toxin(i.e., in a “sandwich” immunoassay). It is also contemplated that thedetection system will be developed as necessary (e.g., the addition ofenzyme substrate in enzyme systems; observation using fluorescent lightfor fluorescent dye systems; and quantitation of radioactivity forradioactive systems).

EXPERIMENTAL

[0177] The following examples serve to illustrate certain preferredembodiments and aspects of the present invention and are not to beconstrued as limiting the scope thereof.

[0178] In the disclosure which follows, the following abbreviationsapply: ° C. (degrees Centigrade); rpm (revolutions per minute);BBS-Tween (borate buffered saline containing Tween); BSA (bovine serumalbumin); ELISA (enzyme-linked immunosorbent assay); CFA (completeFreund's adjuvant); IFA (incomplete Freund's adjuvant); IgG(immunoglobulin G); IgY (immunoglobutin Y); IM (intramuscular); IP(intraperitoneal); IV (intravenous or intravascular); SC (subcutaneous);H₂O (water); HCl (hydrochloric acid); LD₁₀₀ (lethal dose for 100% ofexperimental animals); aa (amino acid); HPLC (high performance liquidchromatography); kD (kilodaltons); gm (grams); μg (micrograms); mg(milligrams); ng (nanograms); μl (microliters); ml (milliliters); mm(millimeters); nm (nanometers); μm (micrometer); M (molar); mM(millimolar); μW (molecular weight); sec (seconds); min(s)(minute/minutes); hr(s) (hour/hours); MgCl₂ (magnesium chloride); NaCl(sodium chloride); Na₂CO₃ (sodium carbonate); OD₂₈₀ (optical density at280 nm); OD₆₀₀ (optical density at 600 nm); PAGE (polyacrylamide gelelectrophoresis); PBS [phosphate buffered saline (150 mM NaCl, 10 mMsodium phosphate buffer, pH 7.2)]; PEG (polyethylene glycol); PMSF(phenylmethylsulfonyl fluoride); SDS (sodium dodecyl sulfate); Tris(tris(hydroxymethyl)aminomethane); Ensure® (Ensure®, Ross Laboratories,Columbus Ohio); Enfamil® (Enfamil®, Mead Johnson); w/v (weight tovolume); v/v (volume to volume); Amicon (Amicon, Inc., Beverly, Mass.);Amresco (Amresco, Inc., Solon, Ohio); ATCC (American Type CultureCollection, Rockville, Md.); BBL (Baltimore Biologics Laboratory, (adivision of Becton Dickinson), Cockeysville, Md.); Becton Dickinson(Becton Dickinson Labware, Lincoln Park, N.J.); BioRad (BioRad,Richmond, Calif.); Biotech (C-C Biotech Corp., Poway, Calif.); CharlesRiver (Charles River Laboratories, Wilmington, Mass.); Cocalico(Cocalico Biologicals Inc., Reamstown, Pa.); CytRx (CytRx Corp.,Norcross, Ga.); Falcon (e.g. Baxter Healthcare Corp., McGaw Park, Ill.and Becton Dickinson); FDA (Federal Food and Drug Administration);Fisher Biotech (Fisher Biotech, Springfield, N.J.); GIBCO (Grand IslandBiologic Company/BRL, Grand Island, N.Y.); Gibco-BRL (Life Technologies,Inc., Gaithersburg, Md.); Harlan Sprague Dawley (Harlan Sprague Dawley,Inc., Madison, Wis.); Mallinckrodt (a division of Baxter HealthcareCorp., McGaw Park, Ill.); Millipore (Millipore Corp., Marlborough,Mass.); New England Biolabs (New England Biolabs, Inc., Beverly, Mass.);Novagen (Novagen, Inc., Madison, Wis.); Pharmacia (Pharmacia, Inc.,Piscataway, N.J.); Qiagen (Qiagen, Chatsworth, Calif.); Sasco (Sasco,Omaha, Nebr.); Showdex (Showa Denko America, Inc., New York, N.Y.);Sigma (Sigma Chemical Co., St. Louis, Mo.); Sterogene (Sterogene, Inc.,Arcadia, Calif.); Tech Lab (Tech Lab, Inc., Blacksburg, Va.); andVaxcell (Vaxcell, Inc., a subsidiary of CytRX Corp., Norcross, Ga.).

[0179] When a recombinant protein is described in the specification itis referred to in a short-hand manner by the amino acids in the toxinsequence present in the recombinant protein rounded to the nearest 10.For example, the recombinant protein pMB1850-2360 contains amino acids1852 through 2362 of the C. difficile toxin B protein. The specificationgives detailed construction details for all recombinant proteins suchthat one skilled in the art will know precisely which amino acids arepresent in a given recombinant protein.

Example 1 Production of High-Titer Antibodies to Clostridium difficileOrganisms in a Hen

[0180] Antibodies to certain pathogenic organisms have been shown to beeffective in treating diseases caused by those organisms. It has notbeen shown whether antibodies can be raised, against Clostridiumdifficile, which would be effective in treating infection by thisorganism. Accordingly, C. difficile was tested as immunogen forproduction of hen antibodies.

[0181] To determine the best course for raising high-titer eggantibodies against whole C. difficile organisms, different immunizingstrains and different immunizing concentrations were examined. Theexample involved (a) preparation of the bacterial immunogen, (b)immunization, (c) purification of anti-bacterial chicken antibodies, and(d) detection of anti-bacterial antibodies in the purified IgYpreparations.

[0182] a) Preparation of Bacterial Immunogen

[0183]C. difficile strains 43594 (serogroup A) and 43596 (serogroup C)were originally obtained from the ATCC. These two strains were selectedbecause they represent two of the most commonly-occurring serogroupsisolated from patients with antibiotic-associated pseudomembranouscolitis. [Delmee et al., J. Clin. Microbiol., 28(10):2210 (1990).]Additionally, both of these strains have been previously characterizedwith respect to their virulence in the Syrian hamster model for C.difficile infection. [Delmee et al., J. Med Microbiol., 33:85 (1990).]

[0184] The bacterial strains were separately cultured on brain heartinfusion agar for 48 hours at 37° C. in a Gas Pack 100 Jar (BBL,Cockeysville, Md.) equipped with a Gas Pack Plus anaerobic envelope(BBL). Forty-eight hour cultures were used because they produce bettergrowth and the organisms have been found to be more cross-reactive withrespect to their surface antigen presentation. The greater the degree ofcross-reactivity of our IgY preparations, the better the probability ofa broad range of activity against different strains/serogroups. [Toma etal., J. Clin. Microbiol., 26(3):426 (1988).]

[0185] The resulting organisms were removed from the agar surface usinga sterile dacron-tip swab, and were suspended in a solution containing0.4% formaldehyde in PBS, pH 7.2. This concentration of formaldehyde hasbeen reported as producing good results for the purpose of preparingwhole-organism immunogen suspensions for the generation of polyclonalanti-C. difficile antisera in rabbits. [Delmee et al., J. Clin.Microbiol., 21:323 (1985); Davies et al., Microbial Path., 9:141(1990).] In this manner, two separate bacterial suspensions wereprepared, one for each strain. The two suspensions were then incubatedat 4° C. for 1 hour. Following this period of formalin-treatment, thesuspensions were centrifuged at 4,200×g for 20 min., and the resultingpellets were washed twice in normal saline. The washed pellets, whichcontained formalin-treated whole organisms, were resuspended in freshnormal saline such that the visual turbidity of each suspensioncorresponded to a #7 McFarland standard. [M. A. C. Edelstein,“Processing Clinical Specimens for Anaerobic Bacteria: Isolation andIdentification Procedures,” in S. M. Finegold et al (eds.)., Bailey andScott's Diagnostic Microbiology, pp. 477-507, C. V. Mosby Co., (1990).The preparation of McFarland nephelometer standards and thecorresponding approximate number of organisms for each tube aredescribed in detail at pp. 172-173 of this volume.] Each of the two #7suspensions was then split into two separate volumes. One volume of eachsuspension was volumetrically adjusted, by the addition of saline, tocorrespond to the visual turbidity of a #1 McFarland standard. [Id.] The#1 suspensions contained approximately 3×10⁸ organisms/ml, and the #7suspensions contained approximately 2×10⁹ organisms/ml. [Id.] The fourresulting concentration-adjusted suspensions of formalin-treated C.difficile organisms were considered to be “bacterial immunogensuspensions.” These suspensions were used immediately after preparationfor the initial immunization. [See section (b).]

[0186] The formalin-treatment procedure did not result in 100%non-viable bacteria in the immunogen suspensions. In order to increasethe level of killing, the formalin concentration and length of treatmentwere both increased for subsequent immunogen preparations, as describedbelow in Table 3. (Although viability was decreased with the strongerformalin treatment, 100% inviability of the bacterial immunogensuspensions was not reached.) Also, in subsequent immunogenpreparations, the formalin solutions were prepared in normal salineinstead of PBS. At day 49, the day of the fifth immunization, the excessvolumes of the four previous bacterial immunogen suspensions were storedfrozen at −70° C. for use during all subsequent immunizations.

[0187] b) Immunization

[0188] For the initial immunization, 1.0 ml volumes of each of the fourbacterial immunogen suspensions described above were separatelyemulsified in 1.2 ml volumes of CFA (GIBCO). For each of the fouremulsified immunogen suspensions, two four-month old White Leghorn hens(pre-laying) were immunized. (It is not necessary to use pre-layinghens; actively-laying hens can also be utilized.) Each hen received atotal volume of approximately 1.0 ml of a single emulsified immunogensuspension via four injections (two subcutaneous and two intramuscular)of approximately 250 μl per site. In this manner, a total of fourdifferent immunization combinations, using two hens per combination,were initiated for the purpose of evaluating both the effect ofimmunizing concentration on egg yolk antibody (IgY) production, andinterstrain cross-reactivity of IgY raised against heterologous strains.The four immunization groups are summarized in Table 3. TABLE 3Immunization Groups Approximate Group Designation Immunizing StrainImmunizing Dose CD 43594, #1 C. difficile 1.5 × 10⁸ organisms/hen strain43594 CD 43594, #7 C. difficile 1.0 × 10⁹ organisms/hen strain 43594 CD43596, #1 C. difficile 1.5 × 10⁸ organisms/hen strain 43596 CD 43596, #7C. difficile 1.0 × 10⁹ organisms/hen strain 43596

[0189] The time point for the first series of immunizations wasdesignated as “day zero.” All subsequent immunizations were performed asdescribed above except that the bacterial immunogen suspensions wereemulsified using IFA (GIBCO) instead of CFA, and for the later timepoint immunization, the stored frozen suspensions were used instead offreshly-prepared suspensions. The immunization schedule used is listedin Table 4. TABLE 4 Immunization Schedule Day Of Immunogen ImmunizationFormalin-Treatment Preparation Used 0 1%,  1 hr. freshly-prepared 14 1%,overnight freshly-prepared 21 1%, overnight freshly-prepared 35 1%, 48hrs. freshly-prepared 49 1%, 72 hrs. freshly-prepared 70 1%, 72 hrs.stored frozen 85 1%, 72 hrs. stored frozen 105 1%, 72 hrs. stored frozen

[0190] c) Purification of Anti-Bacterial Chicken Antibodies

[0191] Groups of four eggs were collected per immunization group betweendays 80 and 84 post-initial immunization, and chicken immunoglobulin(IgY) was extracted according to a modification of the procedure of A.Poison et al., Immunol. Comm., 9:495 (1980). A gentle stream ofdistilled water from a squirt bottle was used to separate the yolks fromthe whites, and the yolks were broken by dropping them through a funnelinto a graduated cylinder. The four individual yolks were pooled foreach group. The pooled, broken yolks were blended with 4 volumes of eggextraction buffer to improve antibody yield (egg extraction buffer is0.01 M sodium phosphate, 0.1 M NaCl, pH 7.5, containing 0.005%thimerosal), and PEG 8000 (Amresco) was added to a concentration of3.5%. When all the PEG dissolved, the protein precipitates that formedwere pelleted by centrifugation at 13,000×g for 10 minutes. Thesupernatants were decanted and filtered through cheesecloth to removethe lipid layer, and the PEG was added to the supernatants to a finalconcentration of 12% (the supernatants were assumed to contain 3.5%PEG). After a second centrifugation, the supernatants were discarded andthe pellets were centrifuged a final time to extrude the remaining PEG.These crude IgY pellets were then dissolved in the original yolk volumeof egg extraction buffer and stored at 4° C. As an additional control, apreimmune IgY solution was prepared as described above, using eggscollected from unimmunized hens.

[0192] d) Detection of Anti-Bacterial Antibodies in the Purified IgYPreparations

[0193] In order to evaluate the relative levels of specific anti-C.difficile activity in the IgY preparations described above, a modifiedversion of the whole-organism ELISA procedure of N. V. Padhye et al., J.Clin. Microbiol. 29:99-103 (1990) was used. Frozen organisms of both C.difficile strains described above were thawed and diluted to aconcentration of approximately 1×10⁷ organisms/ml using PBS, pH 7.2. Inthis way, two separate coating suspensions were prepared, one for eachimmunizing strain. Into the wells of 96-well microtiter plates (Falcon,Pro-Bind Assay Plates) were placed 100 μl volumes of the coatingsuspensions. In this manner, each plate well received a total ofapproximately 1×10⁶ organisms of one strain or the other. The plateswere then incubated at 4° C. overnight. The next morning, the coatingsuspensions were decanted, and all wells were washed three times usingPBS. In order to block non-specific binding sites, 100 μl of 0.5% BSA(Sigma) in PBS was then added to each well, and the plates wereincubated for 2 hours at room temperature. The blocking solution wasdecanted, and 100 μl volumes of the IgY preparations described abovewere initially diluted 1:500 with a solution of 0.1% BSA in PBS, andthen serially diluted in 1:5 steps. The following dilutions were placedin the wells: 1:500, 1:2,500, 1:62,5000, 1:312,500, and 1:1,562,500. Theplates were again incubated for 2 hours at room temperature. Followingthis incubation, the IgY-containing solutions were decanted, and thewells were washed three times using BBS-Tween (0.1 M boric acid, 0.025 Msodium borate, 1.0 M NaCl, 0.1% Tween-20), followed by two washes usingPBS-Tween (0.1% Tween-20), and finally, two washes using PBS only. Toeach well, 100 μl of a 1:750 dilution of rabbit anti-chicken IgG(whole-molecule)-alkaline phosphatase conjugate (Sigma) (diluted in 0.1%BSA in PBS) was added. The plates were again incubated for 2 hours atroom temperature. The conjugate solutions were decanted and the plateswere washed as described above, substituting 50 mM Na₂CO₃, pH 9.5 forthe PBS in the final wash. The plates were developed by the addition of100 μl of a solution containing 1 mg/ml para-nitrophenyl phosphate(Sigma) dissolved in 50 mM Na₂CO₃, 10 mM MgCl₂, pH 9.5 to each well, andincubating the plates at room temperature in the dark for 45 minutes.The absorbance of each well was measured at 410 nm using a Dynatech MR700 plate reader. In this manner, each of the four IgY preparationsdescribed above was tested for reactivity against both of the immunizingC. difficile stains; strain-specific, as well as cross-reactive activitywas determined.

[0194] Table 5 shows the results of the whole-organism ELISA. All fourIgY preparations demonstrated significant levels of activity, to adilution of 1:62,500 or greater against both of the immunizing organismstrains. Therefore, antibodies raised against one strain were highlycross-reactive with the other strain, and vice versa. The immunizingconcentration of organisms did not have a significant effect onorganism-specific IgY production, as both concentrations producedapproximately equivalent responses. Therefore, the lower immunizingconcentration of approximately 1.5×10⁸ organisms/hen is the preferredimmunizing concentration of the two tested. The preimmune IgYpreparation appeared to possess relatively low levels of C.difficile-reactive activity to a dilution of 1:500, probably due toprior exposure of the animals to environmental clostridia.

[0195] An initial whole-organism ELISA was performed using IgYpreparations made from single CD 43594, #1 and CD 43596, #1 eggscollected around day 50 (data not shown). Specific titers were found tobe 5 to 10-fold lower than those reported in Table 5. These resultsdemonstrate that it is possible to begin immunizing hens prior to thetime that they begin to lay eggs, and to obtain high titer specific IgYfrom the first eggs that are laid. In other words, it is not necessaryto wait for the hens to begin laying before the immunization schedule isstarted. TABLE 5 Results Of The Anti-C. difficile Whole-Organism ELISADilution Of 43594- 43596- IgY Preparation IgY Prep Coated Wells CoatedWells CD 43594, #1 1:500 1.746 1.801 1:2,500 1.092 1.670 1:12,500 0.2020.812 1:62,500 0.136 0.179 1:312,500 0.012 0.080 1:1,562,500 0.002 0.020CD 43594, #7 1:500 1.780 1.771 1:2,500 1.025 1.078 1:12,500 0.188 0.3821:62,500 0.052 0.132 1:312,500 0.022 0.043 1:1,562,500 0.005 0.024 CD43596, #1 1:500 1.526 1.790 1:2,500 0.832 1.477 1:12,500 0.247 0.4521:62,500 0.050 0.242 1:312,500 0.010 0.067 1:1,562,500 0.000 0.036 CD43596, #7 1:500 1.702 1.505 1:2,500 0.706 0.866 1:12,500 0.250 0.2821:62,500 0.039 0.078 1:312,500 0.002 0.017 1:1,562,500 0.000 0.010Preimmune IgY 1:500 0.142 0.309 1:2,500 0.032 0.077 1:12,500 0.006 0.0241:62,500 0.002 0.012 1:312,500 0.004 0.010 1:1,562,500 0.002 0.014

Example 2 Treatment of C. difficile Infection with Anti-C. difficileAntibody

[0196] In order to determine whether the immune IgY antibodies raisedagainst whole C. difficile organisms were capable of inhibiting theinfection of hamsters by C. difficile, hamsters infected by thesebacteria were utilized. [Lyerly et al., Infect. Immun., 59:2215-2218(1991).] This example involved: (a) determination of the lethal dose ofC. difficile organisms; and (b) treatment of infected animals withimmune antibody or control antibody in nutritional solution.

[0197] a) Determination of the Lethal Dose of C. diffcile Organisms

[0198] Determination of the lethal dose of C. difficile organisms wascarried out according to the model described by D. M. Lyerly et al.,Infect. Immun., 59:2215-2218 (1991). C. difficile strain ATCC 43596(serogroup C, ATCC) was plated on BHI agar and grown anaerobically (BBLGas Pak 100 system) at 37° C. for 42 hours. Organisms were removed fromthe agar surface using a sterile dacron-tip swab and suspended insterile 0.9% NaCl solution to a density of 10⁸ organisms/ml.

[0199] In order to determine the lethal dose of C. difficile in thepresence of control antibody and nutritional formula, non-immune eggswere obtained from unimmunized hens and a 12% PEG preparation made asdescribed in Example 1(c). This preparation was redissolved in onefourth the original yolk volume of vanilla flavor Ensure®.

[0200] Starting on day one, groups of female Golden Syrian hamsters(Harlan Sprague Dawley), 8-9 weeks old and weighing approximately 100gm, were orally administered 1 ml of the preimmune/Ensure® formula attime zero, 2 hours, 6 hours, and 10 hours. At 1 hour, animals wereorally administered 3.0 mg clindamycin HCl (Sigma) in 1 ml of water.This drug predisposes hamsters to C. difficile infection by altering thenormal intestinal flora. On day two, the animals were given 1 ml of thepreimmune IgY/Ensure® formula at time zero, 2 hours, 6 hours, and 10hours. At 1 hour on day two, different groups of animals were inoculatedorally with saline (control), or 10², 10⁴, 10⁶, or 10⁸ C. difficileorganisms in 1 ml of saline. From days 3-12, animals were given 1 ml ofthe preimmune IgY/Ensure® formula three times daily and observed for theonset of diarrhea and death. Each animal was housed in an individualcage and was offered food and water ad libitum.

[0201] Administration of 10⁶-10⁸ organisms resulted in death in 3-4 dayswhile the lower doses of 10²-10⁴ organisms caused death in 5 days. Cecalswabs taken from dead animals indicated the presence of C. difficile.Given the effectiveness of the 10² dose, this number of organisms waschosen for the following experiment to see if hyperimmune anti-C.difficile antibody could block infection.

[0202] b) Treatment of Infected Animals with Immune Antibody or ControlAntibody in Nutritional Formula

[0203] The experiment in (a) was repeated using three groups of sevenhamsters each. Group A received no clindamycin or C. difficile and wasthe survival control. Group B received clindamycin, 10² C. difficileorganisms and preimmune IgY on the same schedule as the animals in (a)above. Group C received clindamycin, 102 C. difficile organisms, andhyperimmune anti-C. difficile IgY on the same schedule as Group B. Theanti-C. difficile IgY was prepared as described in Example 1 except thatthe 12% PEG preparation was dissolved in one fourth the original yolkvolume of Ensure®.

[0204] All animals were observed for the onset of diarrhea or otherdisease symptoms and death. Each animal was housed in an individual cageand was offered food and water ad libitum. The results are shown inTable 6. TABLE 6 The Effect Of Oral Feeding Of Hyperimmune IgY Antibodyon C. difficile Infection Animal Group Time To Diarrhea^(a) Time ToDeath^(a) A pre-immune IgY only no diarrhea no deaths B Clindamycin, C.difficile, 30 hrs. 49 hrs. preimmune IgY C Clindamycin, C. difficile, 33hrs. 56 hrs. immune IgY

[0205] Hamsters in the control group A did not develop diarrhea andremained healthy during the experimental period. Hamsters in groups Band C developed diarrheal disease. Anti-C. difficile IgY did not protectthe animals from diarrhea or death, all animals succumbed in the sametime interval as the animals treated with preimmune IgY. Thus, whileimmunization with whole organisms apparently can improve sub-lethalsymptoms with particular bacteria (see U.S. Pat. No. 5,080,895 to H.Tokoro), such an approach does not prove to be productive to protectagainst the lethal effects of C. difficile.

Example 3 Production of C. botulinum Type A Antitoxin in Hens

[0206] In order to determine whether antibodies could be raised againstthe toxin produced by clostridial pathogens, which would be effective intreating clostridial diseases, antitoxin to C. botulinum type A toxinwas produced. This example involves: (a) toxin modification; (b)immunization; (c) antitoxin collection; (d) antigenicity assessment; and(e) assay of antitoxin titer.

[0207] a) Toxin Modification

[0208]C. botulinum type A toxoid was obtained from B. R. DasGupta. Fromthis, the active type A neurotoxin (M.W. approximately 150 kD) waspurified to greater than 99% purity, according to published methods. [B.R. DasGupta & V. Sathyamoorthy, Toxicon, 22:415 (1984).] The neurotoxinwas detoxified with formaldehyde according to published methods. [B. R.Singh & B. R. DasGupta, Toxicon, 27:403 (1989).]

[0209] b) Immunization

[0210]C. botulinum toxoid for immunization was dissolved in PBS (1mg/ml) and was emulsified with an approximately equal volume of CFA(GIBCO) for initial immunization or IFA for booster immunization. On dayzero, two white leghorn hens, obtained from local breeders, were eachinjected at multiple sites (intramuscular and subcutaneous) with 1 mlinactivated toxoid emulsified in 1 ml CFA. Subsequent boosterimmunizations were made according to the following schedule for day ofinjection and toxoid amount: days 14 and 21-0.5 mg; day 171-0.75 mg;days 394, 401, 409-0.25 mg. One hen received an additional booster of0.150 mg on day 544.

[0211] c) Antitoxin Collection

[0212] Total yolk immunoglobulin (IgY) was extracted as described inExample 1(c) and the IgY pellet was dissolved in the original yolkvolume of PBS with thimerosal.

[0213] d) Antigenicity Assessment

[0214] Eggs were collected from day 409 through day 423 to assesswhether the toxoid was sufficiently immunogenic to raise antibody. Eggsfrom the two hens were pooled and antibody was collected as described inthe standard PEG protocol. [Example 1(c).] Antigenicity of the botulinaltoxin was assessed on Western blots. The 150 kD detoxified type Aneurotoxin and unmodified, toxic, 300 kD botulinal type A complex (toxinused for intragastric route administration for animal gut neutralizationexperiments; see Example 6) were separated on a SDS-polyacrylamidereducing gel. The Western blot technique was performed according to themethod of Towbin. [H. Towbin et al., Proc. Natl. Acad. Sci. USA, 76:4350(1979).] Ten pg samples of C. botulinum complex and toxoid weredissolved in SDS reducing sample buffer (1% SDS, 0.5% 2-mercaptoethanol,50 mM Tris, pH 6.8, 10% glycerol, 0.025% w/v bromphenol blue, 10%β-mercaptoethanol), heated at 95° C. for 10 min and separated on a 1 mmthick 5% SDS-polyacrylamide gel. [K. Weber and M. Osborn, “Proteins andSodium Dodecyl Sulfate: Molecular Weight Determination on PolyacrylamideGels and Related Procedures,” in The Proteins, 3d Edition (H. Neurath &R. L. Hill, eds), pp. 179-223, (Academic Press, NY, 1975).] Part of thegel was cut off and the proteins were stained with Coomassie Blue. Theproteins in the remainder of the gel were transferred to nitrocelluloseusing the Milliblot-SDE electro-blotting system (Millipore) according tomanufacturer's directions. The nitrocellulose was temporarily stainedwith 10% Ponceau S [S. B. Carroll and A. Laughon, “Production andPurification of Polyclonal Antibodies to the Foreign Segment ofβ-galactosidase Fusion Proteins,” in DNA Cloning. A Practical Approach,Vol.III, (D. Glover, ed.), pp. 89-111, IRL Press, Oxford, (1987)] tovisualize the lanes, then destained by running a gentle stream ofdistilled water over the blot for several minutes. The nitrocellulosewas immersed in PBS containing 3% BSA overnight at 4° C. to block anyremaining protein binding sites.

[0215] The blot was cut into strips and each strip was incubated withthe appropriate primary antibody. The avian anti-C. botulinum antibodies[described in (c)] and pre-immune chicken antibody (as control) werediluted 1:125 in PBS containing 1 mg/ml BSA for 2 hours at roomtemperature. The blots were washed with two changes each of largevolumes of PBS, BBS-Tween and PBS, successively (10 min/wash). Goatanti-chicken IgG alkaline phosphatase conjugated secondary antibody(Fisher Biotech) was diluted 1:500 in PBS containing 1 mg/ml BSA andincubated with the blot for 2 hours at room temperature. The blots werewashed with two changes each of large volumes of PBS and BBS-Tween,followed by one change of PBS and 0.1 M Tris-HCl, pH 9.5. Blots weredeveloped in freshly prepared alkaline phosphatase substrate buffer (100μg/ml nitroblue tetrazolium (Sigma), 50 μg/ml 5-bromo-4-chloro-3-indolylphosphate (Sigma), 5 mM MgCl₂ in 50 mM Na₂CO₃, pH 9.5).

[0216] The Western blots are shown in FIG. 1. The anti-C. botulinum IgYreacted to the toxoid to give a broad immunoreactive band at about145-150 kD on the reducing gel. This toxoid is refractive to disulfidecleavage by reducing agents due to formalin crosslinking. The immune IgYreacted with the active toxin complex, a 97 kD C. botulinum type A heavychain and a 53 kD light chain. The preimmune IgY was unreactive to theC. botulinum complex or toxoid in the Western blot.

[0217] e) Antitoxin Antibody Titer

[0218] The IgY antibody titer to C. botulinum type A toxoid of eggsharvested between day 409 and 423 was evaluated by ELISA, prepared asfollows. Ninety-six-well Falcon Pro-bind plates were coated overnight at4° C. with 100 μl/well toxoid [B. R. Singh & B. R. Das Gupta, Toxicon27:403 (1989)] at 2.5 μg/ml in PBS, pH 7.5 containing 0.005% thimerosal.The following day the wells were blocked with PBS containing 1% BSA for1 hour at 37° C. The IgY from immune or preimmune eggs was diluted inPBS containing 1% BSA and 0.05% Tween 20 and the plates were incubatedfor 1 hour at 37° C. The plates were washed three times with PBScontaining 0.05% Tween 20 and three times with PBS alone. Alkalinephosphatase-conjugated goat-anti-chicken IgG (Fisher Biotech) wasdiluted 1:750 in PBS containing 1% BSA and 0.05% Tween 20, added to theplates, and incubated 1 hour at 37° C. The plates were washed as before,and p-nitrophenyl phosphate (Sigma) at 1 mg/ml in 0.05 M Na₂CO₃, pH 9.5,10 mM MgCl₂ was added.

[0219] The results are shown in FIG. 2. Chickens immunized with thetoxoid generated high titers of antibody to the immunogen. Importantly,eggs from both immunized hens had significant anti-immunogen antibodytiters as compared to preimmune control eggs. The anti-C. botulinum IgYpossessed significant activity, to a dilution of 1:93,750 or greater.

Example 4 Preparation of Avian Egg Yolk Immunoglobulin in an OrallyAdministrable Form

[0220] In order to administer avian IgY antibodies orally toexperimental mice, an effective delivery formula for the IgY had to bedetermined. The concern was that if the crude IgY was dissolved in PBS,the saline in PBS would dehydrate the mice, which might prove harmfulover the duration of the study. Therefore, alternative methods of oraladministration of IgY were tested. The example involved: (a) isolationof immune IgY; (b) solubilization of IgY in water or PBS, includingsubsequent dialysis of the IgY-PBS solution with water to eliminate orreduce the salts (salt and phosphate) in the buffer; and (c) comparisonof the quantity and activity of recovered IgY by absorbance at 280 nmand PAGE, and enzyme-linked immunoassay (ELISA).

[0221] a) Isolation of Immune IgY

[0222] In order to investigate the most effective delivery formula forIgY, we used IgY which was raised against Crotalus durissus terrificusvenom. Three eggs were collected from hens immunized with the C.durissus terrificus venom and IgY was extracted from the yolks using themodified Polson procedure described by Thalley and Carroll[Bio/Technology, 8:934-938 (1990)] as described in Example 1(c).

[0223] The egg yolks were separated from the whites, pooled, and blendedwith four volumes of PBS. Powdered PEG 8000 was added to a concentrationof 3.5%. The mixture was centrifuged at 10,000 rpm for 10 minutes topellet the precipitated protein, and the supernatant was filteredthrough cheesecloth to remove the lipid layer. Powdered PEG 8000 wasadded to the supernatant to bring the final PEG concentration to 12%(assuming a PEG concentration of 3.5% in the supernatant). The 12%PEG/IgY mixture was divided into two equal volumes and centrifuged topellet the IgY.

[0224] b) Solubilization of the IgY in Water or PBS

[0225] One pellet was resuspended in ½ the original yolk volume of PBS,and the other pellet was resuspended in ½ the original yolk volume ofwater. The pellets were then centrifuged to remove any particles orinsoluble material. The IgY in PBS solution dissolved readily but thefraction resuspended in water remained cloudy.

[0226] In order to satisfy anticipated sterility requirements for orallyadministered antibodies, the antibody solution needs to befilter-sterilized (as an alternative to heat sterilization which woulddestroy the antibodies). The preparation of IgY resuspended in water wastoo cloudy to pass through either a 0.2 or 0.45 μm membrane filter, so10 ml of the PBS resuspended fraction was dialyzed overnight at roomtemperature against 250 ml of water. The following morning the dialysischamber was emptied and refilled with 250 ml of fresh H₂O for a seconddialysis. Thereafter, the yields of soluble antibody were determined atOD₂₈₀ and are compared in Table 7. TABLE 7 Dependence Of IgY Yield OnSolvents Absorbance Of 1:10 Percent Fraction Dilution At 280 nm RecoveryPBS dissolved 1.149 100%  H₂O dissolved 0.706 61% PBS dissolved/ 0.88577% H₂O dialyzed

[0227] Resuspending the pellets in PBS followed by dialysis againstwater recovered more antibody than directly resuspending the pellets inwater (77% versus 61%). Equivalent volumes of the IgY preparation in PBSor water were compared by PAGE, and these results were in accordancewith the absorbance values (data not shown).

[0228] c) Activity of IgY Prepared with Different Solvents

[0229] An ELISA was performed to compare the binding activity of the IgYextracted by each procedure described above. C. durissus terrificus(C.d.t.) venom at 2.5 μg/ml in PBS was used to coat each well of a96-well microtiter plate. The remaining protein binding sites wereblocked with PBS containing 5 mg/ml BSA. Primary antibody dilutions (inPBS containing 1 mg/ml BSA) were added in duplicate. After 2 hours ofincubation at room temperature, the unbound primary antibodies wereremoved by washing the wells with PBS, BBS-Tween, and PBS. The speciesspecific secondary antibody (goat anti-chicken immunoglobulinalkaline-phosphatase conjugate (Sigma) was diluted 1:750 in PBScontaining 1 mg/ml BSA and added to each well of the microtiter plate.After 2 hours of incubation at room temperature, the unbound secondaryantibody was removed by washing the plate as before, and freshlyprepared alkaline phosphatase substrate (Sigma) at 1 mg/ml in 50 mMNa₂CO₃, 10 mM MgCl₂, pH 9.5 was added to each well. The colordevelopment was measured on a Dynatech MR 700 microplate reader using a412 nm filter. The results are shown in Table 8.

[0230] The binding assay results parallel the recovery values in Table7, with PBS-dissolved IgY showing slightly more activity than thePBS-dissolved/H₂O dialyzed antibody. The water-dissolved antibody hadconsiderably less binding activity than the other preparations.

Example 5 Survival of Antibody Activity After Passage Through theGastrointestinal Tract

[0231] In order to determine the feasibility of oral administration ofantibody, it was of interest to determine whether orally administeredIgY survived passage through the gastrointestinal tract. The exampleinvolved: (a) oral administration of specific immune antibody mixed witha nutritional formula; and (b) assay of antibody activity extracted fromfeces. TABLE 8 Antigen-Binding Activity Of IgY Prepared With DifferentSolvents Dilution Preimmune PBS Dissolved H₂O Dissolved PBS/H₂O 1:5000.005 1.748 1.577 1.742 1:2,500 0.004 0.644 0.349 0.606 1:12,500 0.0010.144 0.054 0.090 1:62,500 0.001 0.025 0.007 0.016 1:312,500 0.010 0.0000.000 0.002

[0232] a) Oral Administration of Antibody

[0233] The IgY preparations used in this example are the samePBS-dissolved/H₂O dialyzed antivenom materials obtained in Example 4above, mixed with an equal volume of Enfamil®. Two mice were used inthis experiment, each receiving a different diet as follows:

[0234] 1) water and food as usual;

[0235] 2) immune IgY preparation dialyzed against water and mixed 1:1with Enfamil®. (The mice were given the corresponding mixture as theironly source of food and water).

[0236] b) Antibody Activity After Ingestion

[0237] After both mice had ingested their respective fluids, each tubewas refilled with approximately 10 ml of the appropriate fluid firstthing in the morning. By mid-morning there was about 4 to 5 ml of liquidleft in each tube. At this point stool samples were collected from eachmouse, weighed, and dissolved in approximately 500 μl PBS per 100 mgstool sample. One hundred and sixty mg of control stools (no antibody)and 99 mg of experimental stools (specific antibody) in 1.5 ml microfugetubes were dissolved in 800 and 500 μl PBS, respectively. The sampleswere heated at 37° C. for 10 minutes and vortexed vigorously. Theexperimental stools were also broken up with a narrow spatula Eachsample was centrifuged for 5 minutes in a microfuge and thesupernatants, presumably containing the antibody extracts, werecollected. The pellets were saved at 2-8° C. in case future extractswere needed. Because the supernatants were tinted, they were dilutedfive-fold in PBS containing 1 mg/ml BSA for the initial dilution in theenzyme immunoassay (ELISA). The primary extracts were then dilutedfive-fold serially from this initial dilution. The volume of primaryextract added to each well was 190 μl. The ELISA was performed exactlyas described in Example 4. TABLE 9 Specific Antibody Activity AfterPassage Through The Gastrointestinal Tract Control Fecal DilutionPreimmune IgY Extract D EXP. Fecal Extract 1:5 <0 0.000 0.032 1:25 0.016<0 0.016 1:125 <0 <0 0.009 1:625 <0 0.003 0.001 1:3125 <0 <0 0.000

[0238] There was some active antibody in the fecal extract from themouse given the specific antibody in Enfamil® formula, but it waspresent at a very low level. Since the samples were assayed at aninitial 1:5 dilution, the binding observed could have been higher withless dilute samples. Consequently, the mice were allowed to continueingesting either regular food and water or the specific IgY in Enfamil®formula, as appropriate, so the assay could be repeated. Another ELISAplate was coated overnight with 5 μg/ml of C.d.t. venom in PBS.

[0239] The following morning the ELISA plate was blocked with 5 mg/mlBSA, and the fecal samples were extracted as before, except that insteadof heating the extracts at 37° C., the samples were kept on ice to limitproteolysis. The samples were assayed undiluted initially, and in 5×serial dilutions thereafter. Otherwise the assay was carried out asbefore. TABLE 10 Specific Antibody Survives Passage Through TheGastrointestinal Tract Dilution Preimmune IgY Control Extract Exp.Extract undiluted 0.003 <0 0.379 1:5 <0 <0 0.071 1:25 0.000 <0 0.0271:125 0.003 <0 0.017 1:625 0.000 <0 0.008 1:3125 0.002 <0 0.002

[0240] The experiment confirmed the previous results, with the antibodyactivity markedly higher. The control fecal extract showed noanti-C.d.t. activity, even undiluted, while the fecal extract from theanti-C.d.t. IgY/Enfamil®-fed mouse showed considerable anti-C.d.t.activity. This experiment (and the previous experiment) clearlydemonstrate that active IgY antibody survives passage through the mousedigestive tract, a finding with favorable implications for the successof IgY antibodies administered orally as a therapeutic or prophylactic.

Example 6 In Vivo Neutralization of Type C. botulinum Type A Neurotoxinby Avian Antitoxin Antibody

[0241] This example demonstrated the ability of PEG-purified antitoxin,collected as described in Example 3, to neutralize the lethal effect ofC. botulinum neurotoxin type A in mice. To determine the oral lethaldose (LD₁₀₀) of toxin A, groups of BALB/c mice were given differentdoses of toxin per unit body weight (average body weight of 24 grams).For oral administration, toxin A complex, which contains the neurotoxinassociated with other non-toxin proteins was used. This complex ismarkedly more toxic than purified neurotoxin when given by the oralroute. [I. Ohishi et al., Infect. Immun., 16:106 (1977).] C. botulinumtoxin type A complex, obtained from Eric Johnson (University OfWisconsin, Madison) was 250 μg/ml in 50 mM sodium citrate, pH 5.5,specific toxicity 3×10⁷ mouse LD₅₀/mg with parenteral administration.Approximately 40-50 ng/gm body weight was usually fatal within 48 hoursin mice maintained on conventional food and water. When mice were givena diet ad libitum of only Enfamil® the concentration needed to producelethality was approximately 2.5 times higher (125 ng/gm body weight).Botulinal toxin concentrations of approximately 200 ng/gm body weightwere fatal in mice fed Enfamil® containing preimmune IgY (resuspended inEnfamil® at the original yolk volume).

[0242] The oral LD₁₀₀ of C. botulinum toxin was also determined in micethat received known amounts of a mixture of preimmune IgY-Ensure®delivered orally through feeding needles. Using a 22 gauge feedingneedle, mice were given 250 μl each of a preimmune IgY-Ensure® mixture(preimmune IgY dissolved in ¼ original yolk volume) 1 hour before and ½hour and 5 hours after administering botulinal toxin. Toxinconcentrations given orally ranged from approximately 12 to 312 ng/gmbody weight (0.3 to 7.5 μg per mouse). Botulinal toxin complexconcentration of approximately 40 ng/gm body weight (1 μg per mouse) waslethal in all mice in less than 36 hours.

[0243] Two groups of BALB/c mice, 10 per group, were each given orally asingle dose of 1 μg each of botulinal toxin complex in 100 μl of 50 mMsodium citrate pH 5.5. The mice received 250 μl treatments of a mixtureof either preimmune or immune IgY in Ensure® (¼ original yolk volume) 1hour before and ½ hour, 4 hours, and 8 hours after botulinal toxinadministration. The mice received three treatments per day for two moredays. The mice were observed for 96 hours. The survival and mortalityare shown in Table 11. TABLE 11 Neutralization Of Botulinal Toxin A InVivo Toxin Dose Number of Number of ng/gm Antibody Type Mice Alive MiceDead 41.6 non-immune 0 10 41.6 anti-botulinal toxin 10 0

[0244] All mice treated with the preimmune IgY-Ensure® mixture diedwithin 46 hours post-toxin administration. The average time of death inthe mice was 32 hours post toxin administration. Treatments of preimmuneIgY-Ensure® mixture did not continue beyond 24 hours due to extensiveparalysis of the mouth in mice of this group. In contrast, all ten micetreated with the immune anti-botulinal toxin IgY-Ensure® mixturesurvived past 96 hours. Only 4 mice in this group exhibited symptoms ofbotulism toxicity (two mice about 2 days after and two mice 4 days aftertoxin administration). These mice eventually died 5 and 6 days later.Six of the mice in this immune group displayed no adverse effects to thetoxin and remained alive and healthy long term. Thus, the aviananti-botulinal toxin antibody demonstrated very good protection from thelethal effects of the toxin in the experimental mice.

Example 7 Production of an Avian Antitoxin Against Clostridium difficileToxin A

[0245] Toxin A is a potent cytotoxin secreted by pathogenic strains ofC. difficile, that plays a direct role in damaging gastrointestinaltissues. In more severe cases of C. difficile intoxication,pseudomembranous colitis can develop which may be fatal. This would beprevented by neutralizing the effects of this toxin in thegastrointestinal tract. As a first step, antibodies were producedagainst a portion of the toxin. The example involved: (a) conjugation ofa synthetic peptide of toxin A to bovine serum albumin; (b) immunizationof hens with the peptide-BSA conjugate; and (c) detection of antitoxinpeptide antibodies by ELISA.

[0246] a) Conjugation of a Synthetic Peptide of Toxin A to Bovine SerumAlbumin

[0247] The synthetic peptide CQTIDGKKYYFN-NH₂ (SEQ ID NO:82) wasprepared commercially (Multiple Peptide Systems, San Diego, Calif.) andvalidated to be >80% pure by high-pressure liquid chromatography. Theeleven amino acids following the cysteine residue represent a consensussequence of a repeated amino acid sequence found in Toxin A. [Wren etal., Infect. Immun., 59:3151-3155 (1991).] The cysteine was added tofacilitate conjugation to carrier protein.

[0248] In order to prepare the carrier for conjugation, BSA (Sigma) wasdissolved in 0.01 M NAPO₄, pH 7.0 to a final concentration of 20 mg/mland n-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Pierce) wasdissolved in N,N-dimethyl formamide to a concentration of 5 mg/ml. MBSsolution, 0.51 ml, was added to 3.25 ml of the BSA solution andincubated for 30 minutes at room temperature with stirring every 5minutes. The MBS-activated BSA was then purified by chromatography on aBio-Gel P-10 column (Bio-Rad; 40 ml bed volume) equilibrated with 50 mMNaPO₄, pH 7.0 buffer. Peak fractions were pooled (6.0 ml).

[0249] Lyophilized toxin A peptide (20 mg) was added to the activatedBSA mixture, stirred until the peptide dissolved and incubated 3 hoursat room temperature. Within 20 minutes, the reaction mixture becamecloudy and precipitates formed. After 3 hours, the reaction mixture wascentrifuged at 10,000×g for 10 min and the supernatant analyzed forprotein content. No significant protein could be detected at 280 run.The conjugate precipitate was washed three times with PBS and stored at4° C. A second conjugation was performed with 15 mg of activated BSA and5 mg of peptide and the conjugates pooled and suspended at a peptideconcentration of 10 mg/ml in 10 mM NaPO₄, pH 7.2.

[0250] b) Immunization of Hens with Peptide Conjugate

[0251] Two hens were each initially immunized on day zero by injectioninto two subcutaneous and two intramuscular sites with 1 mg of peptideconjugate that was emulsified in CFA (GIBCO). The hens were boosted onday 14 and day 21 with 1 mg of peptide conjugate emulsified in IFA(GIBCO).

[0252] c) Detection of Antitoxin Peptide Antibodies by ELISA

[0253] IgY was purified from two eggs obtained before immunization(pre-immune) and two eggs obtained 31 and 32 days after the initialimmunization using PEG fractionation as described in Example 1.

[0254] Wells of a 96-well microtiter plate (Falcon Pro-Bind Assay Plate)were coated overnight at 4° C. with 100 μg/ml solution of the toxin Asynthetic peptide in PBS, pH 7.2 prepared by dissolving 1 mg of thepeptide in 1.0 ml of H₂O and dilution of PBS. The pre-immune and immuneIgY preparations were diluted in a five-fold series in a buffercontaining 1% PEG 8000 and 0.1% Tween-20 (v/v) in PBS, pH 7.2. The wellswere blocked for 2 hours at room temperature with 150 μl of a solutioncontaining 5% (v/v) Carnation® nonfat dry milk and 1% PEG 8000 in PBS,pH 7.2. After incubation for 2 hours at room temperature, the wells werewashed, secondary rabbit anti-chicken IgG-alkaline phosphatase (1:750)added, the wells washed again and the color development obtained asdescribed in Example 1. The results are shown in Table 12. TABLE 12Reactivity Of IgY With Toxin Peptide Absorbance At 410 nm Dilution ofImmune PEG Prep Preimmune Anti-Peptide 1:100 0.013 0.253 1:500 0.0040.039 1:2500 0.004 0.005

[0255] Clearly, the immune antibodies contain titers against thisrepeated epitope of toxin A.

Example 8 Production of Avian Antitoxins Against Clostridium difficileNative Toxins A and B

[0256] To determine whether avian antibodies are effective for theneutralization of C. difficile toxins, hens were immunized using nativeC. difficile toxins A and B. The resulting egg yolk antibodies were thenextracted and assessed for their ability to neutralize toxins A and B invitro. The Example involved (a) preparation of the toxin immunogens, (b)immunization, (c) purification of the antitoxins, and (d) assay of toxinneutralization activity.

[0257] a) Preparation of the Toxin Immunogens

[0258] Both C. difficile native toxins A and B, and C. difficiletoxoids, prepared by the treatment of the native toxins withformaldehyde, were employed as immunogens. C. difficile toxoids A and Bwere prepared by a procedure which was modified from published methods(Ehrich et al., Infect. Immun. 28:1041 (1980). Separate solutions (inPBS) of native C. difficile toxin A and toxin B (Tech Lab) were eachadjusted to a concentration of 0.20 mg/ml, and formaldehyde was added toa final concentration of 0.4%. The toxin/formaldehyde solutions werethen incubated at 37° C. for 40 hrs. Free formaldehyde was then removedfrom the resulting toxoid solutions by dialysis against PBS at 4° C. Inpreviously published reports, this dialysis step was not performed.Therefore, free formaldehyde must have been present in their toxoidpreparations. The toxoid solutions were concentrated, using a Centriprepconcentrator unit (Amicon), to a final toxoid concentration of 4.0mg/ml. The two resulting preparations were designated as toxoid A andtoxoid B.

[0259]C. difficile native toxins were prepared by concentrating stocksolutions of toxin A and toxin B (Tech Lab, Inc), using Centriprepconcentrator units (Amicon), to a final concentration of 4.0 mg/ml.

[0260] b) Immunization

[0261] The first two immunizations were performed using the toxoid A andtoxoid B immunogens described above. A total of 3 different immunizationcombinations were employed. For the first immunization group, 0.2 ml oftoxoid A was emulsified in an equal volume of Titer Max adjuvant(CytRx). Titer Max was used in order to conserve the amount of immunogenused, and to simplify the immunization procedure. This immunizationgroup was designated “CTA.” For the second immunization group, 0.1 ml oftoxoid B was emulsified in an equal volume of Titer Max adjuvant. Thisgroup was designated “CTB.” For the third immunization group, 0.2 ml oftoxoid A was first mixed with 0.2 ml of toxoid B, and the resultingmixture was emulsified in 0.4 ml of Titer Max adjuvant. This group wasdesignated “CTAB.” In this way, three separate immunogen emulsions wereprepared, with each emulsion containing a final concentration of 2.0mg/ml of toxoid A (CTA) or toxoid B (CTB) or a mixture of 2.0 mg/mltoxoid A and 2.0 mg/ml toxoid B (CTAB).

[0262] On day 0, White Leghorn hens, obtained from a local breeder, wereimmunized as follows: Group CTA. Four hens were immunized, with each henreceiving 200 μg of toxoid A, via two intramuscular (I.M.) injections of50 μl of CTA emulsion in the breast area. Group CTB. One hen wasimmunized with 200 μg of toxoid B, via two I.M. injections of 50 μl ofCTB emulsion in the breast area. Group CTAB. Four hens were immunized,with each hen receiving a mixture containing 200 μg of toxoid A and 200μg of toxoid B, via two I.M. injections of 100 μl of CTAB emulsion inthe breast area. The second immunization was performed 5 weeks later, onday 35, exactly as described for the first immunization above.

[0263] In order to determine whether hens previously immunized with C.difficile toxoids could tolerate subsequent booster immunizations usingnative toxins, a single hen from group CTAB was immunized for a thirdtime, this time using a mixture of the native toxin A and native toxin Bdescribed in section (a) above (these toxins were notformaldehyde-treated, and were used in their active form). This was donein order to increase the amount (titer) and affinity of specificantitoxin antibody produced by the hen over that achieved by immunizingwith toxoids only. On day 62, 0.1 ml of a toxin mixture was preparedwhich contained 200 μg of native toxin A and 200 μg of native toxin B.This toxin mixture was then emulsified in 0.1 ml of Titer Max adjuvant.A single CTAB hen was then immunized with the resulting immunogenemulsion, via two I.M. injections of 100 μl each, into the breast area.This hen was marked with a wing band, and observed for adverse effectsfor a period of approximately 1 week, after which time the hen appearedto be in good health.

[0264] Because the CTAB hen described above tolerated the boosterimmunization with native toxins A and B with no adverse effects, it wasdecided to boost the remaining hens with native toxin as well. On day70, booster immunizations were performed as follows: Group CTA. A 0.2 mlvolume of the 4 mg/ml native toxin A solution was emulsified in an equalvolume of Titer Max adjuvant. Each of the 4 hens was then immunized with200 μg of native toxin A, as described for the toxoid A immunizationsabove. Group CTB. A 50 μl volume of the 4 mg/ml native toxin B solutionwas emulsified in an equal volume of Titer Max adjuvant. The hen wasthen immunized with 200 μg of native toxin B, as described for thetoxoid B immunizations above. Group CTAB. A 0.15 ml volume of the 4mg/ml native toxin A solution was first mixed with a 0.15 ml volume the4 mg/ml native toxin B solution. The resulting toxin mixture was thenemulsified in 0.3 ml of Titer Max adjuvant. The 3 remaining hens (thehen with the wing band was not immunized this time) were then immunizedwith 200 μg of native toxin A and 200 μg of native toxin B as describedfor the toxoid A+ toxoid B immunizations (CTAB) above. On day 85, allhens received a second booster immunization using native toxins, doneexactly as described for the first boost with native toxins above.

[0265] All hens tolerated both booster immunizations with native toxinswith no adverse effects. As previous literature references describe theuse of formaldehyde-treated toxoids, this is apparently the first timethat any immunizations have been performed using native C. difficiletoxins.

[0266] c) Purification of Antitoxins

[0267] Eggs were collected from the hen in group CTB 10-12 daysfollowing the second immunization with toxoid (day 35 immunizationdescribed in section. (b) above), and from the hens in groups CTA andCTAB 20-21 days following the second immunization with toxoid. To beused as a pre-immune (negative) control, eggs were also collected fromunimmunized hens from the same flock. Egg yolk immunoglobulin (IgY) wasextracted from the 4 groups of eggs as described in Example 1(c), andthe final IgY pellets were solubilized in the original yolk volume ofPBS without thimerosal. Importantly, thimerosal was excluded because itwould have been toxic to the CHO cells used in the toxin neutralizationassays described in section (d) below.

[0268] d) Assay of Toxin Neutralization Activity

[0269] The toxin neutralization activity of the IgY solutions preparedin section (c) above was determined using an assay system that wasmodified from published methods. [Ehrich et al., Infect. Immun.28:1041-1043 (1992); and McGee et al. Microb. Path. 12:333-341 (1992).]As additional controls, affinity-purified goat anti-C. difficile toxin A(Tech Lab) and affinity-purified goat anti-C. difficile toxin B (TechLab) were also assayed for toxin neutralization activity.

[0270] The IgY solutions and goat antibodies were serially diluted usingF 12 medium (GIBCO) which was supplemented with 2% FCS (GIBCO)(thissolution will be referred to as “medium” for the remainder of thisExample). The resulting antibody solutions were then mixed with astandardized concentration of either native C. difficile toxin A (TechLab), or native C. difficile toxin B (Tech Lab), at the concentrationsindicated below. Following incubation at 37° C. for 60 min., 100 μlvolumes of the toxin+antibody mixtures were added to the wells of96-well microtiter plates (Falcon Microtest III) which contained 2.5×10⁴Chinese Hamster Ovary (CHO) cells per well (the CHO cells were plated onthe previous day to allow them to adhere to the plate wells). The finalconcentration of toxin, or dilution of antibody indicated below refersto the final test concentration of each reagent present in therespective microtiter plate wells. Toxin reference wells were preparedwhich contained CHO cells and toxin A or toxin B at the sameconcentration used for the toxin plus antibody mixtures (these wellscontained no antibody). Separate control wells were also prepared whichcontained CHO cells and medium only. The assay plates were thenincubated for 18-24 hrs. in a 37° C., humidified, 5% CO₂ incubator. Onthe following day, the remaining adherent (viable) cells in the platewells were stained using 0.2% crystal violet (Mallinckrodt) dissolved in2% ethanol, for 10 min. Excess stain was then removed by rinsing withwater, and the stained cells were solubilized by adding 100 μl of 1% SDS(dissolved in water) to each well. The absorbance of each well was thenmeasured at 570 nm, and the percent cytotoxicity of each test sample ormixture was calculated using the following formula:${\% \quad {CHO}\quad {Cell}\quad {Cytotoxicity}} = {\left\lbrack {1 - \left( \frac{{Abs}.\quad {Sample}}{{Abs}.\quad {Control}} \right)} \right\rbrack \times 100}$

[0271] Unlike previous reports which quantitate results visually bycounting cell rounding by microscopy, this Example utilizedspectrophotometric methods to quantitate the C. difficile toxinbioassay. In order to determine the toxin A neutralizing activity of theCTA, CTAB, and pre-immune IgY preparations, as well as theaffinity-purified goat antitoxin A control, dilutions of theseantibodies were reacted against a 0.1 μg/ml concentration of nativetoxin A (this is the approx. 50% cytotoxic dose of toxin A in this assaysystem). The results are shown in FIG. 3.

[0272] Complete neutralization of toxin A occurred with the CTA IgY(antitoxin A, above) at dilutions of 1:80 and lower, while significantneutralization occurred out to the 1:320 dilution. The CTAB IgY(antitoxin A+toxin B, above) demonstrated complete neutralization at the1:320-1:160 and lower dilutions, and significant neutralization occurredout to the 1:1280 dilution. The commercially available affinity-purifiedgoat antitoxin A did not completely neutralize toxin A at any of thedilutions tested, but demonstrated significant neutralization out to adilution of 1:1,280. The preimmune IgY did not show any toxin Aneutralizing activity at any of the concentrations tested. These resultsdemonstrate that IgY purified from eggs laid by hens immunized withtoxin A alone, or simultaneously with toxin A and toxin B, is aneffective toxin A antitoxin.

[0273] The toxin B neutralizing activity of the CTAB and pre-immune IgYpreparations, and also the affinity-purified goat antitoxin B controlwas determined by reacting dilutions of these antibodies against aconcentration of native toxin B of 0.1 ng/ml (approximately the 50%cytotoxic dose of toxin B in the assay system). The results are shown inFIG. 4.

[0274] Complete neutralization of toxin B occurred with the CTAB IgY(antitoxin A+toxin B, above) at the 1:40 and lower dilutions, andsignificant neutralization occurred out to the 1:320 dilution. Theaffinity-purified goat antitoxin B demonstrated complete neutralizationat dilutions of 1:640 and lower, and significant neutralization occurredout to a dilution of 1:2,560. The preimmune IgY did not show any toxin Bneutralizing activity at any of the concentrations tested. These resultsdemonstrate that IgY purified from eggs laid by hens immunizedsimultaneously with toxin A and toxin B is an effective toxin Bantitoxin.

[0275] In a separate study, the toxin B neutralizing activity of CTB,CTAB, and pre-immune IgY preparations was determined by reactingdilutions of these antibodies against a native toxin B concentration of0.1 μg/ml (approximately 100% cytotoxic dose of toxin B in this assaysystem). The results are shown in FIG. 5.

[0276] Significant neutralization of toxin B occurred with the CTB IgY(antitoxin B, above) at dilutions of 1:80 and lower, while the CTAB IgY(antitoxin A+toxin B, above) was found to have significant neutralizingactivity at dilutions of 1:40 and lower. The preimmune IgY did not showany toxin B neutralizing activity at any of the concentrations tested.These results demonstrate that IgY purified from eggs laid by hensimmunized with toxin B alone, or simultaneously with toxin A and toxinB, is an effective toxin B antitoxin.

Example 9 In Vivo Protection of Golden Syrian Hamsters From C. difficileDisease by Avian Antitoxins Against C. difficile Toxins A and B

[0277] The most extensively used animal model to study C. difficiledisease is the hamster. [Lyerly et al., Infect. Immun. 47:349-352(1992).] Several other animal models for antibiotic-induced diarrheaexist, but none mimic the human form of the disease as closely as thehamster model. [R. Fekety, “Animal Models of Antibiotic-InducedColitis,” in O. Zak and M. Sande (eds.), Experimental Models inAntimicrobial Chemotherapy, Vol. 2, pp.61-72, (1986).] In this model,the animals are first predisposed to the disease by the oraladministration of an antibiotic, such as clindamycin, which alters thepopulation of normally-occurring gastrointestinal flora (Fekety, at61-72). Following the oral challenge of these animals with viable C.difficile organisms, the hamsters develop cecitis, and hemorrhage,ulceration, and inflammation are evident in the intestinal mucosa.[Lyerly et al., Infect. Immun. 47:349-352 (1985).] The animals becomelethargic, develop severe diarrhea, and a high percentage of them diefrom the disease. [Lyerly et al. Infect. Immun. 47:349-352 (1985).] Thismodel is therefore ideally suited for the evaluation of therapeuticagents designed for the treatment or prophylaxis of C. difficiledisease.

[0278] The ability of the avian C. difficile antitoxins, described inExample 1 above, to protect hamsters from C. difficile disease wasevaluated using the Golden Syrian hamster model of C. difficileinfection. The Example involved (a) preparation of the avian C.difficile antitoxins, (b) in vivo protection of hamsters from C.difficile disease by treatment with avian antitoxins, and (c) long-termsurvival of treated hamsters.

[0279] a) Preparation of the Avian C. difficile Antitoxins

[0280] Eggs were collected from hens in groups CTA and CTAB described inExample 1(b) above. To be used as a pre-immune (negative) control, eggswere also purchased from a local supermarket. Egg yolk immunoglobulin(IgY) was extracted from the 3 groups of eggs as described in Example1(c), and the final IgY pellets were solubilized in one fourth theoriginal yolk volume of Ensure® nutritional formula.

[0281] b) In Vivo Protection of Hamsters Against C. difficile Disease byTreatment with Avian Antitoxins

[0282] The avian C. difficile antitoxins prepared in section (a) abovewere evaluated for their ability to protect hamsters from C. difficiledisease using an animal model system which was modified from publishedprocedures. [Fekety, at 61-72; Borriello et al., J. Med. Microbiol.,24:53-64 (1987); Kim et al., Infect. Immun., 55:2984-2992 (1987);Borriello et al., J. Med. Microbiol., 25:191-196 (1988); Delmee andAvesani, J. Med. Microbiol., 33:85-90 (1990); and Lyerly et al., Infect.Immun., 59:2215-2218 (1991).] For the study, three separate experimentalgroups were used, with each group consisting of 7 female Golden Syrianhamsters (Charles River), approximately 10 weeks old and weighingapproximately 100 gms. each. The three groups were designated “CTA,”“CTAB” and “Pre-immune.” These designations corresponded to theantitoxin preparations with which the animals in each group weretreated. Each animal was housed in an individual cage, and was offeredfood and water ad libitum through the entire length of the study. On day1, each animal was orally administered 1.0 ml of one of the threeantitoxin preparations (prepared in section (a) above) at the followingtimepoints: 0 hrs., 4 hrs., and 8 hrs. On day 2, the day 1 treatment wasrepeated. On day 3, at the 0 hr. timepoint, each animal was againadministered antitoxin, as described above. At 1 hr., each animal wasorally administered 3.0 mg of clindamycin-HCl (Sigma) in 1 ml of water.This treatment predisposed the animals to infection with C. difficile.As a control for possible endogenous C. difficile colonization, anadditional animal from the same shipment (untreated) was alsoadministered 3.0 mg of clindamycin-HCl in the same manner. Thisclindamycin control animal was left untreated (and uninfected) for theremainder of the study. At the 4 hr. and 8 hr. timepoints, the animalswere administered antitoxin as described above. On day 4, at the 0 hr.timepoint, each animal was again administered antitoxin as describedabove. At 1 hr., each animal was orally challenged with 1 ml of C.difficile inoculum, which contained approx. 100 C. difficile strain43596 organisms in sterile saline. C. difficile strain 43596, which is aserogroup C strain, was chosen because it is representative of one ofthe most frequently-occurring serogroups isolated from patients withantibiotic-associated pseudomembranous colitis. [Delmee et al., J. Clin.Microbiol., 28:2210-2214 (1985).] In addition, this strain has beenpreviously demonstrated to be virulent in the hamster model ofinfection. [Delmee and Avesani, J. Med. Microbiol., 33:85-90 (1990).] Atthe 4 hr. and 8 hr. timepoints, the animals were administered antitoxinas described above. On days 5 through 13, the animals were administeredantitoxin 3× per day as described for day 1 above, and observed for theonset of diarrhea and death. On the morning of day 14, the final resultsof the study were tabulated. These results are shown in Table 13.

[0283] Representative animals from those that died in the Pre-Immune andCTA groups were necropsied. Viable C. difficile organisms were culturedfrom the ceca of these animals, and the gross pathology of thegastrointestinal tracts of these animals was consistent with thatexpected for C. difficile disease (inflamed, distended, hemorrhagiccecum, filled with watery diarrhea-like material). In addition, theclindamycin control animal remained healthy throughout the entire studyperiod, therefore indicating that the hamsters used in the study had notpreviously been colonized with endogenous C. difficile organisms priorto the start of the study. Following the final antitoxin treatment onday 13, a single surviving animal from the CTA group, and also from theCTAB group, was sacrificed and necropsied. No pathology was noted ineither animal. TABLE 13 Treatment Results No. No. Animals AnimalsTreatment Group Surviving Dead Pre-Immune 1 6 CTA (Antitoxin A only) 5 2CTAB (Antitoxin A + 7 0 Antitoxin B)

[0284] Treatment of hamsters with orally-administered toxin A and toxinB antitoxin (group CTAB) successfully protected 7 out of 7 (100%) of theanimals from C. difficile disease. Treatment of hamsters withorally-administered toxin A antitoxin (group CTA) protected 5 out of 7(71%) of these animals from C. difficile disease. Treatment usingpre-immune IgY was not protective against C. difficile disease, as only1 out of 7 (14%) of these animals survived. These results demonstratethat the avian toxin A antitoxin and the avian toxin A+toxin B antitoxineffectively protected the hamsters from C. difficile disease. Theseresults also suggest that although the neutralization of toxin A aloneconfers some degree of protection against C. difficile disease, in orderto achieve maximal protection, simultaneous antitoxin A and antitoxin Bactivity is necessary.

[0285] c) Long-Term Survival of Treated Hamsters

[0286] It has been previously reported in the literature that hamsterstreated with orally-administered bovine antitoxin IgG concentrate areprotected from C. difficile disease as long as the treatment iscontinued, but when the treatment is stopped, the animals developdiarrhea and subsequently die within 72 hrs. [Lyerly et al., Infect.Immun., 59(6):2215-2218 (1991).]

[0287] In order to determine whether treatment of C. difficile diseaseusing avian antitoxins promotes long-term survival following thediscontinuation of treatment, the 4 surviving animals in group CTA, andthe 6 surviving animals in group CTAB were observed for a period of 11days (264 hrs.) following the discontinuation of antitoxin treatmentdescribed in section (b) above. All hamsters remained healthy throughthe entire post-treatment period. This result demonstrates that not onlydoes treatment with avian antitoxin protect against the onset of C.difficile disease (i.e., it is effective as a prophylactic), it alsopromotes long-term survival beyond the treatment period, and thusprovides a lasting cure.

Example 10 In Vivo Treatment of Established C. difficile Infection inGolden Syrian Hamsters with Avian Antitoxins Against C. difficile ToxinsA and B

[0288] The ability of the avian C. difficile antitoxins, described inExample 8 above, to treat an established C. difficile infection wasevaluated using the Golden Syrian hamster model. The Example involved(a) preparation of the avian C. difficile antitoxins, (b) in vivotreatment of hamsters with established C. difficile infection, and (c)histologic evaluation of cecal tissue.

[0289] a) Preparation of the Avian C. difficile Antitoxins

[0290] Eggs were collected from hens in group CTAB described in Example8 (b) above, which were immunized with C. difficile toxoids and nativetoxins A and B. Eggs purchased from a local supermarket were used as apre-immune (negative) control. Egg yolk immunoglobulin (IgY) wasextracted from the 2 groups of eggs as described in Example 1(c), andthe final IgY pellets were solubilized in one-fourth the original yolkvolume of Ensure® nutritional formula.

[0291] b) In Vivo Treatment of Hamsters with Established C. difficileInfection

[0292] The avian C. difficile antitoxins prepared in section (a) abovewere evaluated for the ability to treat established C. difficileinfection in hamsters using an animal model system which was modifiedfrom the procedure which was described for the hamster protection studyin Example 8(b) above.

[0293] For the study, four separate experimental groups were used, witheach group consisting of 7 female Golden Syrian hamsters (CharlesRiver), approx. 10 weeks old, weighing approximately 100 gms. each. Eachanimal was housed separately, and was offered food and water ad libitumthrough the entire length of the study.

[0294] On day 1 of the study, the animals in all four groups were eachpredisposed to C. difficile infection by the oral administration of 3.0mg of clindamycin-HCl (Sigma) in 1 ml of water.

[0295] On day 2, each animal in all four groups was orally challengedwith 1 ml of C. difficile inoculum, which contained approximately 100 C.difficile strain 43596 organisms in sterile saline. C. difficile strain43596 was chosen because it is representative of one of the mostfrequently-occurring serogroups isolated from patients withantibiotic-associated pseudomembranous colitis. [Delmee et al., J. Clin.Microbiol., 28:2210-2214 (1990).] In addition, as this was the same C.difficile strain used in all of the previous Examples above, it wasagain used in order to provide experimental continuity.

[0296] On day 3 of the study (24 hrs. post-infection), treatment wasstarted for two of the four groups of animals. Each animal of one groupwas orally administered 1.0 ml of the CTAB IgY preparation (prepared insection (a) above) at the following timepoints: 0 hrs., 4 hrs., and 8hrs. The animals in this group were designated “CTAB-24.” The animals inthe second group were each orally administered 1.0 ml of the pre-immuneIgY preparation (also prepared in section (a) above) at the sametimepoints as for the CTAB group. These animals were designated“Pre-24.” Nothing was done to the remaining two groups of animals on day3.

[0297] On day 4, 48 hrs. post-infection, the treatment described for day3 above was repeated for the CTAB-24 and Pre-24 groups, and wasinitiated for the remaining two groups at the same timepoints. The finaltwo groups of animals were designated “CTAB-48” and “Pre-48”respectively.

[0298] On days 5 through 9, the animals in all four groups wereadministered antitoxin or pre-immune IgY, 3× per day, as described forday 4 above. The four experimental groups are summarized in Table 14.TABLE 14 Experimental Treatment Groups Group Designation ExperimentalTreatment CTAB-24 Infected, treatment w/antitoxin IgY started @ 24 hrs.post-infection. Pre-24 Infected, treatment w/pre-immune IgY started @ 24hrs. post-infection. CTAB-48 Infected, treatment w/antitoxin IgY started@ 48 hrs. post-infection. Pre-48 Infected, treatment w/pre-immune IgYstarted @ 48 hrs. post-infection.

[0299] All animals were observed for the onset of diarrhea and deaththrough the conclusion of the study on the morning of day 10. Theresults of this study are displayed in Table 15. TABLE 15 ExperimentalOutcome--Day 10 No. No. Treatment Animals Animals Group Surviving DeadCTAB-24 6 1 Pre-24 0 7 CTAB-48 4 3 Pre-48 2 5

[0300] Eighty-six percent of the animals which began receiving treatmentwith antitoxin IgY at 24 hrs. post-infection (CTAB-24 above) survived,while 57% of the animals treated with antitoxin IgY starting 48 hrs.post-infection (CTAB-48 above) survived. In contrast, none of theanimals receiving pre-immune IgY starting 24 hrs. post-infection (Pre-24above) survived, and only 29% of the animals which began receivingtreatment with pre-immune IgY at 48 hrs. post-infection (Pre-48 above)survived through the conclusion of the study. These results demonstratethat avian antitoxins raised against C. difficile toxins A and B arecapable of successfully treating established C. difficile infections invivo.

[0301] c) Histologic Evaluation of Cecal Tissue

[0302] In order to further evaluate the ability of the IgY preparationstested in this study to treat established C. difficile infection,histologic evaluations were performed on cecal tissue specimens obtainedfrom representative animals from the study described in section (b)above.

[0303] Immediately following death, cecal tissue specimens were removedfrom animals which died in the Pre-24 and Pre-48 groups. Following thecompletion of the study, a representative surviving animal wassacrificed and cecal tissue specimens were removed from the CTAB-24 andCTAB-48 groups. A single untreated animal from the same shipment asthose used in the study was also sacrificed and a cecal tissue specimenwas removed as a normal control. All tissue specimens were fixedovernight at 4° C. in 10% buffered formalin. The fixed tissues wereparaffin-embedded, sectioned, and mounted on glass microscope slides.The tissue sections were then stained using hematoxylin and eosin (H andE stain), and were examined by light microscopy.

[0304] Upon examination, the tissues obtained from the CTAB-24 andCTAB-48 animals showed no pathology, and were indistinguishable from thenormal control. This observation provides further evidence for theability of avian antitoxins raised against C. difficile toxins A and Bto effectively treat established C. difficile infection, and to preventthe pathologic consequences which normally occur as a result of C.difficile disease.

[0305] In contrast, characteristic substantial mucosal damage anddestruction was observed in the tissues of the animals from the Pre-24and Pre-48 groups which died from C. difficile disease. Normal tissuearchitecture was obliterated in these two preparations, as most of themucosal layer was observed to have sloughed away, and there werenumerous large hemorrhagic areas containing massive numbers oferythrocytes.

Example 11 Cloning and Expression of C. difficile Toxin A Fragments

[0306] The toxin A gene has been cloned and sequenced, and shown toencode a protein of predicted MW of 308 kd. [Dove et al., Infect.Immun., 58:480-488 (1990).] Given the expense and difficulty ofisolating native toxin A protein, it would be advantageous to use simpleand inexpensive procaryotic expression systems to produce and purifyhigh levels of recombinant toxin A protein for immunization purposes.Ideally, the isolated recombinant protein would be soluble in order topreserve native antigenicity, since solubilized inclusion body proteinsoften do not fold into native conformations. To allow ease ofpurification, the recombinant protein should be expressed to levelsgreater than 1 mg/liter of E. coli culture.

[0307] To determine whether high levels of recombinant toxin A proteincan be produced in E. coli, fragments of the toxin A gene were clonedinto various prokaryotic expression vectors, and assessed for theability to express recombinant toxin A protein in E. coli. Threeprokaryotic expression systems were utilized. These systems were chosenbecause they drive expression of either fusion (pMALc and pGEX2T) ornative (pET23a-c) protein to high levels in E. coli, and allow affinitypurification of the expressed protein on a ligand containing column.Fusion proteins expressed from pGEX vectors bind glutathione agarosebeads, and are eluted with reduced glutathione. pMAL fusion proteinsbind amylose resin, and are eluted with maltose. A poly-histidine tag ispresent at either the N-terminal (pET16b) or C-terminal (pET23a-c) endof pET fusion proteins. This sequence specifically binds Ni₂ ⁺ chelatecolumns, and is eluted with imidazole salts. Extensive descriptions ofthese vectors are available [Williams et al. (1995) DNA Cloning 2:Expression Systems, Glover and Hames, eds. IRL Press, Oxford, pp.15-58], and will not be discussed in detail here. The Example involved(a) cloning of the toxin A gene, (b) expression of large fragments oftoxin A in various prokaryotic expression systems, (c) identification ofsmaller toxin A gene fragments that express efficiently in E. coli, (d)purification of recombinant toxin A protein by affinity chromatography,and (e) demonstration of functional activity of a recombinant fragmentof the toxin A gene.

[0308] a) Cloning of the Toxin A Gene

[0309] A restriction map of the toxin A gene is shown in FIG. 6. Theencoded protein contains a carboxy terminal ligand binding region,containing multiple repeats of a carbohydrate binding domain. [vonEichel-Streiber and Sauerbom, Gene 96:107-113 (1990).] The toxin A genewas cloned in three pieces, by using either the polymerase chainreaction (PCR) to amplify specific regions, (regions 1 and 2, FIG. 6) orby screening a constructed genomic library for a specific toxin A genefragment (region 3, FIG. 6). The sequences of the utilized PCR primersare P1: 5′ GGAAATT TAGCTGCAGCATCTGAC 3′ (SEQ ID NO.: 1); P2: 5′TCTAGCAAATTCGCTTGT GTTGAA 3′ (SEQ ID NO.:2); P3: 5′ CTCGCATATAGCATTAGACC3′ (SEQ ID NO.:3); and P4: 5′ CTATCTAGGCCTAAAGTAT 3′ (SEQ ID NO.:4).These regions were cloned into prokaryotic expression vectors thatexpress either fusion (pMALc and pGEX2T) or native (pET23a-c) protein tohigh levels in E. coli, and allow affinity purification of the expressedprotein on a ligand containing column.

[0310]Clostridium difficile VPI strain 10463 was obtained from the ATCC(ATCC #43255) and grown under anaerobic conditions in brain-heartinfusion medium (BBL). High molecular-weight C. difficile DNA wasisolated essentially as described by Wren and Tabaqchali (1987) J. Clin.Microbiol., 25:2402, except proteinase K and sodium dodecyl sulfate(SDS) was used to disrupt the bacteria, and cetyltrimethylammoniumbromide precipitation [as described in Ausubel et al., Current Protocolsin Molecular Biology (1989)] was used to remove carbohydrates from thecleared lysate. The integrity and yield of genomic DNA was assessed bycomparison with a serial dilution of uncut lambda DNA afterelectrophoresis on an agarose gel.

[0311] Fragments 1 and 2 were cloned by PCR, utilizing a proofreadingthermostable DNA polymerase (native pfu polymerase; Stratagene). Thehigh fidelity of this polymerase reduces the mutation problemsassociated with amplification by error prone polymerases (e.g., Taqpolymerase). PCR amplification was performed using the indicated PCRprimers (FIG. 6) in 50 μl reactions containing 10 mM Tris-HCl (8.3), 50mM KCl, 1.5 mM MgCl₂, 200 μM each dNTP, 0.2 μM each primer, and 50 ng C.difficile genomic DNA. Reactions were overlaid with 100 μl mineral oil,heated to 94° C. for 4 min, 0.5 μl native pfu polymerase (Stratagene)added, and the reaction cycled 30× at 94° C. for 1 min, 50° C. for 1min, 72° C. for 4 min, followed by 10 min at 72° C. Duplicate reactionswere pooled, chloroform extracted, and ethanol precipitated. Afterwashing in 70% ethanol, the pellets were resuspended in 50 μl TE buffer[10 mM Tris-HCL, 1 mM EDTA pH 8.0]. Aliquots of 10 μl each wererestriction digested with either EcoRI/HincII (fragment 1) or EcoRI/PstI(fragment 2), and the appropriate restriction fragments were gelpurified using the Prep-A-Gene kit (BioRad), and ligated to eitherEcoRI/SmaI-restricted pGEX2T (Pharmacia) vector (fragment 1), or theEcoRI/PstI pMA1c (New England Biolabs) vector (fragment 2). Both clonesare predicted to produce in-frame fusions with either theglutathione-S-transferase protein (pGEX vector) or the maltose bindingprotein (pMAL vector). Recombinant clones were isolated, and confirmedby restriction digestion, using standard recombinant molecular biologytechniques. [Sambrook et al., Molecular Cloning, A Laboratory Manual(1989), and designated pGA30-660 and pMA660-1100, respectively (see FIG.6 for description of the clone designations).]

[0312] Fragment 3 was cloned from a genomic library of size selectedPstI digested C. difficile genomic DNA, using standard molecular biologytechniques (Sambrook et al.). Given that the fragment 3 internal PstIsite is protected from cleavage in C. difficile genomic DNA [Price etal., Curr. Microbiol., 16:55-60 (1987)], a 4.7 kb fragment from PstIrestricted C. difficile genomic DNA was gel purified, and ligated toPstI restricted, phosphatase treated pUC9 DNA. The resulting genomiclibrary was screened with a oligonucleotide primer specific to fragment3, and multiple independent clones were isolated. The presence offragment 3 in several of these clones was confirmed by restrictiondigestion, and a clone of the indicated orientation (FIG. 6) wasrestricted with BamHI/HindIII, the released fragment purified by gelelectrophoresis, and ligated into similarly restricted pET23c expressionvector DNA (Novagen). Recombinant clones were isolated, and confirmed byrestriction digestion. This construct is predicted to create both apredicted in frame fusion with the pET protein leader sequence, as wellas a predicted C-terminal poly-histidine affinity tag, and is designatedpPA1100-2680 (see FIG. 6 for the clone designation).

[0313] b) Expression of Large Fragments of Toxin A in E. coli

[0314] Protein expression from the three expression constructs made in(a) was induced, and analyzed by Western blot analysis with an affinitypurified, goat polyclonal antiserum directed against the toxin A toxoid(Tech Lab). The procedures utilized for protein induction, SDS-PAGE, andWestern blot analysis are described in detail in Williams et al (1995),supra. In brief, 5 ml 2×YT (16 g tryptone, 10 g yeast extract, 5 g NaClper liter, pH 7.5+100 μg/ml ampicillin were added to cultures ofbacteria (BL21 for pMA1 and pGEX plasmids, and BL21(DE3)LysS for pETplasmids) containing the appropriate recombinant clone which wereinduced to express recombinant protein by addition of IPTG to 1 mM.Cultures were grown at 37° C., and induced when the cell density reached0.5 OD₆₀₀. Induced protein was allowed to accumulate for two hrs afterinduction. Protein samples were prepared by pelleting 1 ml aliquots ofbacteria by centrifugation (1 min in a microfuge), and resuspension ofthe pelleted bacteria in 150 μl of 2×SDS-PAGE sample buffer [Williams etal. (1995), supra]. The samples were heated to 95° C. for 5 min, thecooled and 5 or 10 μl aliquots loaded on 7.5% SDS-PAGE gels. BioRad highmolecular weight protein markers were also loaded, to allow estimationof the MW of identified fusion proteins. After electrophoresis, proteinwas detected either generally by staining gels with Coomassie blue, orspecifically, by blotting to nitrocellulose for Western blot detectionof specific immunoreactive protein. Western blots, (performed asdescribed in Example 3) which detect toxin A reactive protein in celllysates of induced protein from the three expression constructs areshown in FIG. 7. In this figure, lanes 1-3 contain cell lysates preparedfrom E. coli strains containing pPA1100-2860 in B 121(DE3)LysE cells;lanes 4-6 contain cell lysates prepared from E. coli strains containingpPA1100-2860 in B121(DE3)LysS cells; lanes 7-9 contain cell lysatesprepared from E. coli strains containing pMA30-660; lanes 10-12 containcell lysates prepared from E. coli strains containing pMA660-1100. Thelanes were probed with an affinity purified goat antitoxin A polyclonalantibody (Tech Lab). Control lysates from uninduced cells (lanes 1, 7,and 10) contain very little immunoreactive material compared to theinduced samples in the remaining lanes. The highest molecular weightband observed for each clone is consistent with the predicted size ofthe full length fusion protein.

[0315] Each construct directs expression of high molecular weight (HMW)protein that is reactive with the toxin A antibody. The size of thelargest immunoreactive bands from each sample is consistent withpredictions of the estimated MW of the intact fusion proteins. Thisdemonstrates that the three fusions are in-frame, and that none of theclones contain cloning artifacts that disrupt the integrity of theencoded fusion protein. However, the Western blot demonstrates thatfusion protein from the two larger constructs (pGA30-660 andpPA1100-2680) are highly degraded. Also, expression levels of toxin Aproteins from these two constructs are low, since induced protein bandsare not visible by Coomassie staining (not shown). Several otherexpression constructs that fuse large sub-regions of the toxin A gene toeither pMALc or pET23a-c expression vectors, were constructed and testedfor protein induction. These constructs were made by mixing gel purifiedrestriction fragments, derived from the expression constructs shown inFIG. 6, with appropriately cleaved expression vectors, ligating, andselecting recombinant clones in which the toxin A restriction fragmentshad ligated together and into the expression vector as predicted forin-frame fusions. The expressed toxin A interval within these constructsare shown in FIG. 8, as well as the internal restriction sites utilizedto make these constructs.

[0316] As used herein, the term “interval” refers to any portion (i.e.,any segment of the toxin which is less than the whole toxin molecule) ofa clostridial toxin. In a preferred embodiment, “interval” refers toportions of C. difficile toxins such as toxin A or toxin B. It is alsocontemplated that these intervals will correspond to epitopes ofimmunologic importance, such as antigens or immunogens against which aneutralizing antibody response is effected. It is not intended that thepresent invention be limited to the particular intervals or sequencesdescribed in these Examples. It is also contemplated that sub-portionsof intervals (e.g., an epitope contained within one interval or whichbridges multiple intervals) be used as compositions and in the methodsof the present invention.

[0317] In all cases, Western blot analysis of each of these constructswith goat antitoxin A antibody (Tech Lab) detected HMW fusion protein ofthe predicted size (not shown). This confirms that the reading frame ofeach of these clones is not prematurely terminated, and is fused in thecorrect frame with the fusion partner. However, the Western blotanalysis revealed that in all cases, the induced protein is highlydegraded, and, as assessed by the absence of identifiable inducedprotein bands by Coomassie Blue staining, are expressed only at lowlevels. These results suggest that expression of high levels of intacttoxin A recombinant protein is not possible when large regions of thetoxin A gene are expressed in E. coli using these expression vectors.

[0318] c) High Level Expression of Small Toxin A Protein Fusions in E.coli

[0319] Experience indicates that expression difficulties are oftenencountered when large (greater than 100 kd) fragments are expressed inE. coli. A number of expression constructs containing smaller fragmentsof the toxin A gene were constructed, to determine if small regions ofthe gene can be expressed to high levels without extensive proteindegradation. A summary of these expression constructs are shown in FIG.9. All were constructed by in-frame fusions of convenient toxin Arestriction fragments to either the pMALc or pET23a-c vectors. Proteinpreparations from induced cultures of each of these constructs wereanalyzed by both Coomassie Blue staining and Western analysis as in (b)above. In all cases, higher levels of intact, full length fusionproteins were observed than with the larger recombinants from section(b).

[0320] d) Purification of Recombinant Toxin A Protein

[0321] Large scale (500 ml) cultures of each recombinant from (c) weregrown, induced, and soluble and insoluble protein fractions wereisolated. The soluble protein extracts were affinity chromatographed toisolate recombinant fusion protein, as described [Williams et al.(1994), supra]. In brief, extracts containing tagged pET fusions werechromatographed on a nickel chelate column, and eluted using imidazolesalts as described by the distributor (Novagen). Extracts containingsoluble pMAL fusion protein were prepared and chromatographed in columnbuffer (10 mM NaPO₄, 0.5M NaCl, 10 mM β-mercaptoethanol, pH 7.2) over anamylose resin column (New England Biolabs), and eluted with columnbuffer containing 10 mM maltose as described [Williams et al. (1995),supra]. When the expressed protein was found to be predominantlyinsoluble, insoluble protein extracts were prepared by the methoddescribed in Example 17, infra. The results are summarized in Table 16.FIG. 10 shows the sample purifications of recombinant toxin A protein.In this figure, lanes 1 and 2 contain MBP fusion protein purified byaffinity purification of soluble protein. TABLE 16 Purification OfRecombinant Toxin A Protein Yield Affinity % Intact Yield Intact ProteinPurified Soluble Soluble Fusion Insoluble Fusion Clone ^((a)) SolubilityProtein ^((b)) Protein ^((c)) Protein pMA30-270 Soluble 4 mg/500 mls 10%NA PMA30-300 Soluble 4 mg/500 mls 5-10%  NA pMA300-660 Insoluble — NA 10mg/500 ml pMA660-1100 Soluble 4.5 mg/500 mls 50% NA pMA1100-1610 Soluble18 mg/500 mls 10% NA pMA1610-1870 Both 22 mg/500 mls 90% 20 mg/500 mlpMA1450-1870 Insoluble — NA 0.2 mg/500 ml pPA1100-1450 Soluble 0.1mg/500 mls 90% NA pPA1100-1870 Soluble 0.02 mg/500 mls 90% NApMA1870-2680 Both 12 mg/500 mls 80% NA pPa1870-2680 Insoluble — NA 10mg/500 ml

[0322] Lanes 3 and 4 contain MBP fusion protein purified bysolubilization of insoluble inclusion bodies. The purified fusionprotein samples are pMA1870-2680 (lane 1), pMA660-1100 (lane 2),pMA300-600 (lane 3) and pMA1450-1870 (lane 4).

[0323] Poor yields of affinity purified protein were obtained whenpoly-histidine tagged pET vectors were used to drive expression(pPA1100-1450, pP1100-1870). However, significant protein yields wereobtained from pMAL expression constructs spanning the entire toxin Agene, and yields of full-length soluble fusion protein ranged from anestimated 200-400 μg/500 ml culture (pMA30-300) to greater than 20mg/500 ml culture (pMA1610-1870). Only one interval was expressed tohigh levels as strictly insoluble protein (pMA300-660). Thus, althoughhigh level expression was not observed when using large expressionconstructs from the toxin A gene, usable levels of recombinant proteinspanning the entire toxin A gene were obtainable by isolating inducedprotein from a series of smaller pMAL expression constructs that spanthe entire toxin A gene. This is the first demonstration of thefeasibility of expressing recombinant toxin A protein to high levels inE. coli.

[0324] e) Hemagglutination Assay Using the Toxin A Recombinant Proteins

[0325] The carboxy terminal end consisting of the repeating unitscontains the hemagglutination activity or binding domain of C. difficiletoxin A. To determine whether the expressed toxin A recombinants retainfunctional activity, hemagglutination assays were performed. Two toxin Arecombinant proteins, one containing the binding domain as eithersoluble affinity purified protein (pMA1870-2680) or SDS solubilizedinclusion body protein (pPA1870-2680) and soluble protein from oneregion outside that domain (pMA1100-1610) were tested using a describedprocedure. [H. C. Krivan et. al., Infect. Immun., 53:573 (1986).]Citrated rabbit red blood cells (RRBC)(Cocalico) were washed severaltimes with Tris-buffer (0.1M Tris and 50 mM NaCl) by centrifugation at450×g for 10 minutes at 4° C. A 1% RRBC suspension was made from thepacked cells and resuspended in Tris-buffer. Dilutions of therecombinant proteins and native toxin A (Tech Labs) were made in theTris-buffer and added in duplicate to a round-bottomed 96-wellmicrotiter plate in a final volume of 100 μl. To each well, 50 μl of the1% RRBC suspension was added, mixed by gentle tapping, and incubated at4° C. for 3-4 hours. Significant hemagglutination occurred only in therecombinant proteins containing the binding domain (pMA 1870-2680) andnative toxin A. The recombinant protein outside the binding domain (pMA1100-1610) displayed no hemagglutination activity. Using equivalentprotein concentrations, the hemagglutination titer for toxin A was1:256, while titers for the soluble and insoluble recombinant proteinsof the binding domain were 1:256 and about 1:5000. Clearly, therecombinant proteins tested retained functional activity and were ableto bind RRBC's.

Example 12 Functional Activity of IgY Reactive Against Toxin ARecombinants

[0326] The expression of recombinant toxin A protein as multiplefragments in E. coli has demonstrated the feasibility of generatingtoxin A antigen through use of recombinant methodologies (Example 11).The isolation of these recombinant proteins allows the immunoreactivityof each individual subregion of the toxin A protein to be determined(i.e., in a antibody pool directed against the native toxin A protein).This identifies the regions (if any) for which little or no antibodyresponse is elicited when the whole protein is used as a immunogen.Antibodies directed against specific fragments of the toxin A proteincan be purified by affinity chromatography against recombinant toxin Aprotein, and tested for neutralization ability. This identifies anytoxin A subregions that are essential for producing neutralizingantibodies. Comparison with the levels of immune response directedagainst these intervals when native toxin is used as an immunogenpredicts whether potentially higher titers of neutralizing antibodiescan be produced by using recombinant protein directed against aindividual region, rather than the entire protein. Finally, since it isunknown whether antibodies reactive to the recombinant toxin A proteinsproduced in Example 11 neutralize toxin A as effectively as antibodiesraised against native toxin A (Examples 9 and 10), the protectiveability of a pool of antibodies affinity purified against recombinanttoxin A fragments was assessed for its ability to neutralize toxin A.

[0327] This Example involved (a) epitope mapping of the toxin A proteinto determine the titre of specific antibodies directed againstindividual subregions of the toxin A protein when native toxin A proteinis used as an immunogen, (b) affinity purification of IgY reactiveagainst recombinant proteins spanning the toxin A gene, (c) toxin Aneutralization assays with affinity purified IgY reactive to recombinanttoxin A protein to identify subregions of the toxin A protein thatinduce the production of neutralizing antibodies, and determination ofwhether complete neutralization of toxin A can be elicited with amixture of antibodies reactive to recombinant toxin A protein.

[0328] a) Epitope Mapping of the Toxin A Gene

[0329] The affinity purification of recombinant toxin A protein specificto defined intervals of the toxin A protein allows epitope mapping ofantibody pools directed against native toxin A. This has not previouslybeen possible, since previous expression of toxin A recombinants hasbeen assessed only by Western blot analysis, without knowledge of theexpression levels of the protein [e.g., von Eichel-Streiber et al, J.Gen. Microbiol., 135:55-64 (1989)]. Thus, high or low reactivity ofrecombinant toxin A protein on Western blots may reflect proteinexpression level differences, not immunoreactivity differences. Giventhat the purified recombinant protein generated in Example 11 have beenquantitated, the issue of relative immunoreactivity of individualregions of the toxin A protein was precisely addressed.

[0330] For the purposes of this Example, the toxin A protein wassubdivided into 6 intervals (1-6), numbered from the amino (interval 1)to the carboxyl (interval 6) termini.

[0331] The recombinant proteins corresponding to these intervals werefrom expression clones (see Example 11(d) for clone designations)pMA30-300 (interval 1), pMA300-660 (interval 2), pMA660-1100 (interval3), pPA1100-1450 (interval 4), pMA1450-1870 (interval 5) andpMA1870-2680 (interval 6). These 6 clones were selected because theyspan the entire protein from amino acids numbered 30 through 2680, andsubdivide the protein into 6 small intervals. Also, the carbohydratebinding repeat interval is contained specifically in one interval(interval 6), allowing evaluation of the immune response specificallydirected against this region. Western blots of 7.5% SDS-PAGE gels,loaded and electrophoresed with defined quantities of each recombinantprotein, were probed with either goat antitoxin A polyclonal antibody(Tech Lab) or chicken antitoxin A polyclonal antibody [pCTA IgY, Example8(c)]. The blots were prepared and developed with alkaline phosphataseas previously described [Williams et al. (1995), supra]. At least 90% ofall reactivity, in either goat or chicken antibody pools, was found tobe directed against the ligand binding domain (interval 6). Theremaining immunoreactivity was directed against all five remainingintervals, and was similar in both antibody pools, except that thechicken antibody showed a much lower reactivity against interval 2 thanthe goat antibody.

[0332] This clearly demonstrates that when native toxin A is used as animmunogen in goats or chickens, the bulk of the immune response isdirected against the ligand binding domain of the protein, with theremaining response distributed throughout the remaining ⅔ of theprotein.

[0333] b) Affinity Purification of IgY Reactive Against RecombinantToxin A Protein

[0334] Affinity columns, containing recombinant toxin A protein from the6 defined intervals in (a) above, were made and used to (i) affinitypurify antibodies reactive to each individual interval from the CTA IgYpreparation [Example 8(c)], and (ii) deplete interval specificantibodies from the CTA IgY preparation. Affinity columns were made bycoupling 1 ml of PBS-washed Actigel resin (Sterogene) with regionspecific protein and {fraction (1/10)} final volume of Ald-couplingsolution (1M sodium cyanoborohydride). The total region specific proteinadded to each reaction mixture was 2.7 mg (interval 1), 3 mg (intervals2 and 3), 0.1 mg (interval 4), 0.2 mg (interval 5) and 4 mg (interval6). Protein for intervals 1, 3, and 6 was affinity purified pMA1 fusionprotein in column buffer (see Example 11). Interval 4 was affinitypurified poly-histidine containing pET fusion in PBS; intervals 2 and 5were from inclusion body preparations of insoluble pMAL fusion protein,dialyzed extensively in PBS. Aliquots of the supernatants from thecoupling reactions, before and after coupling, were assessed byCoomassie staining of 7.5% SDS-PAGE gels. Based on protein bandintensities, in all cases greater than 50% coupling efficiencies wereestimated. The resins were poured into 5 ml BioRad columns, washedextensively with PBS, and stored at 4° C.

[0335] Aliquots of the CTA IgY polyclonal antibody preparation weredepleted for each individual region as described below. A 20 ml sampleof the CTA IgY preparation [Example 8(c)] was dialyzed extensivelyagainst 3 changes of PBS (1 liter for each dialysis), quantitated byabsorbance at OD₂₈₀, and stored at 4° C. Six 1 ml aliquots of thedialyzed IgY preparation were removed, and depleted individually foreach of the six intervals. Each 1 ml aliquot was passed over theappropriate affinity column, and the eluate twice reapplied to thecolumn. The eluate was collected, and pooled with a 1 ml PBS wash. Boundantibody was eluted from the column by washing with 5 column volumes of4 M Guanidine-HCl (in 10 mM Tris-HCl, pH 8.0). The column wasreequilibrated in PBS, and the depleted antibody stock reapplied asdescribed above. The eluate was collected, pooled with a 1 ml PBS wash,quantitated by absorbance at OD₂₈₀, and stored at 4° C. In this manner,6 aliquots of the CTA IgY preparation were individually depleted foreach of the 6 toxin A intervals, by two rounds of affinity depletion.The specificity of each depleted stock was tested by Western blotanalysis. Multiple 7.5% SDS-PAGE gels were loaded with protein samplescorresponding to all 6 toxin A subregions. After electrophoresis, thegels were blotted, and protein transfer confirmed by Ponceau S staining[protocols described in Williams et al. (1995), supra]. After blockingthe blots 1 hr at 20° C. in PBS+0.1% Tween 20 (PBST) containing 5% milk(as a blocking buffer), 4 ml of either a {fraction (1/500)} dilution ofthe dialyzed CTA IgY preparation in blocking buffer, or an equivalentamount of the six depleted antibody stocks (using OD₂₈₀ to standardizeantibody concentration) were added and the blots incubated a further 1hr at room temperature. The blots were washed and developed withalkaline phosphatase (using a rabbit anti-chicken alkaline phosphateconjugate as a secondary antibody) as previously described [Williams etal. (1995), supra]. In all cases, only the target interval was depletedfor antibody reactivity, and at least 90% of the reactivity to thetarget intervals was specifically depleted.

[0336] Region specific antibody pools were isolated by affinitychromatography as described below. Ten mls of the dialyzed CTA IgYpreparation were applied sequentially to each affinity column, such thata single 10 ml aliquot was used to isolate region specific antibodiesspecific to each of the six subregions. The columns were sequentiallywashed with 10 volumes of PBS, 6 volumes of BBS-Tween, 10 volumes ofTBS, and eluted with 4 ml Actisep elution media (Sterogene). The eluatewas dialyzed extensively against several changes of PBS, and theaffinity purified antibody collected and stored at 4° C. The volumes ofthe eluate increased to greater than 10 mls during dialysis in eachcase, due to the high viscosity of the Actisep elution media. Aliquotsof each sample were 20× concentrated using Centricon 30microconcentrators (Amicon) and stored at 4° C. The specificity of eachregion specific antibody pool was tested, relative to the dialyzed CTAIgY preparation, by Western blot analysis, exactly as described above,except that 4 ml samples of blocking buffer containing 100 μl regionspecific antibody (unconcentrated) were used instead of the depleted CTAIgY preparations. Each affinity purified antibody preparation wasspecific to the defined interval, except that samples purified againstintervals 1-5 also reacted with interval 6. This may be due tonon-specific binding to the interval 6 protein, since this proteincontains the repetitive ligand binding domain which has been shown tobind antibodies nonspecifically. [Lyerly et al., Curr. Microbiol.,19:303-306 (1989).]

[0337] The reactivity of each affinity purified antibody preparation tothe corresponding proteins was approximately the same as the reactivityof the {fraction (1/500)} diluted dialyzed CTA IgY preparation standard.Given that the specific antibody stocks were diluted {fraction (1/40)},this would indicate that the unconcentrated affinity purified antibodystocks contain {fraction (1/10)}-{fraction (1/20)} the concentration ofspecific antibodies relative to the starting CTA IgY preparation.

[0338] c) Toxin A Neutralization Assay Using Antibodies Reactive TowardRecombinant Toxin A Protein

[0339] The CHO toxin neutralization assay [Example 8(d)] was used toassess the ability of the depleted or enriched samples generated in (b)above to neutralize the cytotoxicity of toxin A. The general ability ofaffinity purified antibodies to neutralize toxin A was assessed bymixing together aliquots of all 6 concentrated stocks of the 6 affinitypurified samples generated in (b) above, and testing the ability of thismixture to neutralize a toxin A concentration of 0.1 μg/ml. The results,shown in FIG. 11, demonstrate almost complete neutralization of toxin Ausing the affinity purified (AP) mix. Some epitopes within therecombinant proteins utilized for affinity purification were probablylost when the proteins were denatured before affinity purification [byGuanidine-HCl treatment in (b) above]. Thus, the neutralization abilityof antibodies directed against recombinant protein is probablyunderestimated using these affinity purified antibody pools. Thisexperiment demonstrates that antibodies reactive to recombinant toxin Acan neutralize cytotoxicity, suggesting that neutralizing antibodies maybe generated by using recombinant toxin A protein as immunogen.

[0340] In view of the observation that the recombinant expression clonesof the toxin A gene divide the protein into 6 subregions, theneutralizing ability of antibodies directed against each individualregion was assessed. The neutralizing ability of antibodies directedagainst the ligand binding domain of toxin A was determined first.

[0341] In the toxin neutralization experiment shown in FIG. 11, interval6 specific antibodies (interval 6 contains the ligand binding domain)were depleted from the dialyzed PEG preparation, and the effect on toxinneutralization assayed. Interval 6 antibodies were depleted either byutilizing the interval 6 depleted CTA IgY preparation from (b) above(“−6 aff. depleted” in FIG. 11), or by addition of interval 6 protein tothe CTA IgY preparation (estimated to be a 10 fold molar excess overanti-interval 6 immunoglobulin present in this preparation) tocompetitively compete for interval 6 protein (“−6 prot depleted” in FIG.11). In both instances, removal of interval 6 specific antibodiesreduces the neutralization efficiency relative to the starting CTA IgYpreparation. This demonstrates that antibodies directed against interval6 contribute to toxin neutralization. Since interval 6 corresponds tothe ligand binding domain of the protein, these results demonstrate thatantibodies directed against this region in the PEG preparationcontribute to the neutralization of toxin A in this assay. However, itis significant that after removal of these antibodies, the PEGpreparation retains significant ability to neutralize toxin A (FIG. 11).This neutralization is probably due to the action of antibodies specificto other regions of the toxin A protein, since at least 90% of theligand binding region reactive antibodies were removed in the depletedsample prepared in (b) above. This conclusion was supported bycomparison of the toxin neutralization of the affinity purified (AP) mixcompared to affinity purified interval 6 antibody alone. Although someneutralization ability was observed with AP interval 6 antibodies alone,the neutralization was significantly less than that observed with themixture of all 6 AP antibody stocks (not shown).

[0342] Given that the mix of all six affinity purified samples almostcompletely neutralized the cytotoxicity of toxin A (FIG. 11), therelative importance of antibodies directed against toxin A intervals 1-5within the mixture was determined. This was assessed in two ways. First,samples containing affinity purified antibodies representing 5 of the 6intervals were prepared, such that each individual region was depletedfrom one sample. FIG. 12 demonstrates a sample neutralization curve,comparing the neutralization ability of affinity purified antibody mixeswithout interval 4 (−4) or 5 (−5) specific antibodies, relative to themix of all 6 affinity purified antibody stocks (positive control). Whilethe removal of interval 5 specific antibodies had no effect on toxinneutralization (or intervals 1-3, not shown), the loss of interval 4specific antibodies significantly reduced toxin neutralization (FIG.12).

[0343] Similar results were seen in a second experiment, in whichaffinity purified antibodies, directed against a single region, wereadded to interval 6 specific antibodies, and the effects on toxinneutralization assessed. Only interval 4 specific antibodiessignificantly enhanced neutralization when added to interval 6 specificantibodies (FIG. 13). These results demonstrate that antibodies directedagainst interval 4 (corresponding to clone pPA1100-1450 in FIG. 9) areimportant for neutralization of cytotoxicity in this assay. Epitopemapping has shown that only low levels of antibodies reactive to thisregion are generated when native toxin A is used as an immunogen[Example 12(a)]. It is hypothesized that immunization with recombinantprotein specific to this interval will elicit higher titers ofneutralizing antibodies. In summary, this analysis has identified twocritical regions of the toxin A protein against which neutralizingantibodies are produced, as assayed by the CHO neutralization assay.

Example 13 Production and Evaluation of Avian Antitoxin Against C.difficile Recombinant Toxin A Polypeptide

[0344] In Example 12, we demonstrated neutralization of toxin A mediatedcytotoxicity by affinity purified antibodies reactive to recombinanttoxin A protein. To determine whether antibodies raised against arecombinant polypeptide fragment of C. difficile toxin A may beeffective in treating clostridial diseases, antibodies to recombinanttoxin A protein representing the binding domain were generated. Twotoxin A binding domain recombinant polypeptides, expressing the bindingdomain in either the pMALc (pMA1870-2680) or pET 23(pPA1870-2680)vector, were used as immunogens. The pMAL protein was affinity purifiedas a soluble product [Example 12(d)] and the pET protein was isolated asinsoluble inclusion bodies [Example 12(d)] and solubilized to animmunologically active protein using a proprietary method described in apending patent application (U.S. patent application Ser. No.08/129,027). This Example involves (a) immunization, (b) antitoxincollection, (c) determination of antitoxin antibody titer, (d)anti-recombinant toxin A neutralization of toxin A hemagglutinationactivity in vitro, and (e) assay of in vitro toxin A neutralizingactivity.

[0345] a) Immunization

[0346] The soluble and the inclusion body preparations each were usedseparately to immunize hens. Both purified toxin A polypeptides werediluted in PBS and emulsified with approximately equal volumes of CFAfor the initial immunization or IFA for subsequent boosterimmunizations. On day zero, for each of the recombinant preparations,two egg laying white Leghorn hens (obtained from local breeder) wereeach injected at multiple sites (intramuscular and subcutaneous) with 1ml of recombinant adjuvant mixture containing approximately 0.5 to 1.5mgs of recombinant toxin A. Booster immunizations of 1.0 mg were givenon days 14 and day 28.

[0347] b) Antitoxin Collection

[0348] Total yolk immune IgY was extracted as described in the standardPEG protocol (as in Example 1) and the final IgY pellet was dissolved insterile PBS at the original yolk volume. This material is designated“immune recombinant IgY” or “immune IgY.”

[0349] c) Antitoxin Antibody Titer

[0350] To determine if the recombinant toxin A protein was sufficientlyimmunogenic to raise antibodies in hens, the antibody titer of arecombinant toxin A polypeptide was determined by ELISA. Eggs from bothhens were collected on day 32, the yolks pooled and the antibody wasisolated using PEG as described. The immune recombinant IgY antibodytiter was determined for the soluble recombinant protein containing themaltose binding protein fusion generated in p-Mal (pMA1870-2680).Ninety-six well Falcon Pro-bind plates were coated overnight at 4° C.with 100 μl/well of toxin A recombinant at 2.5 μg/μl in PBS containing0.05% thimerosal. Another plate was also coated with maltose bindingprotein (MBP) at the same concentration, to permit comparison ofantibody reactivity to the fusion partner. The next day, the wells wereblocked with PBS containing 1% bovine serum albumin (BSA) for 1 hour at37° C. IgY isolated from immune or preimmune eggs was diluted inantibody diluent (PBS containing 1% BSA and 0.05% Tween-20), and addedto the blocked wells and incubated for 1 hour at 37° C. The plates werewashed three times with PBS with 0.05% Tween-20, then three times withPBS. Alkaline phosphatase conjugated rabbit anti-chicken IgG (Sigma)diluted 1:1000 in antibody diluent was added to the plate, and incubatedfor 1 hour at 37° C. The plates were washed as before and substrate wasadded, [p-nitrophenyl phosphate (Sigma)] at 1 mg/ml in 0.05M Na₂CO₃, pH9.5 and 10 mM MgCl₂. The plates were evaluated quantitatively on aDynatech MR 300 Micro EPA plate reader at 410 nm about 10 minutes afterthe addition of substrate.

[0351] Based on these ELISA results, high antibody titers were raised inchickens immunized with the toxin A recombinant polypeptide. Therecombinant appeared to be highly immunogenic, as it was able togenerate high antibody titers relatively quickly with few immunizations.Immune IgY titer directed specifically to the toxin A portion of therecombinant was higher than the immune IgY titer to its fusion partner,the maltose binding protein, and significantly higher than the preimmuneIgY. ELISA titers (reciprocal of the highest dilution of IgY generatinga signal) in the preimmune IgY to the MBP or the recombinant was <1:30while the immune IgY titers to MBP and the toxin A recombinant were1:18750 and >1:93750 respectively. Importantly, the anti-recombinantantibody titers generated in the hens against the recombinantpolypeptide is much higher, compared to antibodies to that region raisedusing native toxin A. The recombinant antibody titer to region 1870-2680in the CTA antibody preparation is at least five-fold lower compared tothe recombinant generated antibodies (1:18750 versus >1:93750). Thus, itappears a better immune response can be generated against a specificrecombinant using that recombinant as the immunogen compared to thenative toxin A.

[0352] This observation is significant, as it shows that becauserecombinant portions stimulate the production of antibodies, it is notnecessary to use native toxin molecules to produce antitoxinpreparations. Thus, the problems associated with the toxicity of thenative toxin are avoided and large-scale antitoxin production isfacilitated.

[0353] d) Anti-Recombinant Toxin A Neutralization of Toxin AHemagglutination Activity In Vitro

[0354] Toxin A has hemagglutinating activity besides cytotoxic andenterotoxin properties. Specifically, toxin A agglutinates rabbiterythrocytes by binding to a trisaccharide (gal 1-3B 1-4GlcNAc) on thecell surface. [H. Krivan et al., Infect. Immun., 53:573-581 (1986).] Weexamined whether the anti-recombinant toxin A (immune IgY, antibodiesraised against the insoluble product expressed in pET) can neutralizethe hemagglutination activity of toxin A in vitro. The hemagglutinationassay procedure used was described by H. C. Krivan et al. Polyethyleneglycol-fractionated immune or preimmune IgY were pre-absorbed withcitrated rabbit erythrocytes prior to performing the hemagglutinationassay because we have found that IgY alone can agglutinate red bloodcells. Citrated rabbit red blood cells (RRBC's) (Cocalico) were washedtwice by centrifugation at 450×g with isotonic buffer (0.1 M Tris-HCl,0.05 M NaCl, pH 7.2). RRBC-reactive antibodies in the IgY were removedby preparing a 10% RRBC suspension (made by adding packed cells toimmune or preimmune IgY) and incubating the mixture for 1 hour at 37° C.The RRBCs were then removed by centrifugation. Neutralization of thehemagglutination activity of toxin A by antibody was tested inround-bottomed 96-well microtiter plates. Twenty-five μl of toxin A (36μg/ml) (Tech Lab) in isotonic buffer was mixed with an equal volume ofdifferent dilutions of immune or preimmune IgY in isotonic buffer, andincubated for 15 minutes at room temperature. Then, 50 μl of a 1% RRBCsuspension in isotonic buffer was added and the mixture was incubatedfor 3 hours at 4° C. Positive control wells containing the finalconcentration of 9 μg/ml of toxin A after dilution without IgY were alsoincluded. Hemagglutination activity was assessed visually, with adiffuse matrix of RRBC's coating the bottom of the well representing apositive hemagglutination reaction and a tight button of RRBC's at thebottom of the well representing a negative reaction. Theanti-recombinant immune IgY neutralized toxin A hemagglutinationactivity, giving a neutralization titer of 1:8. However, preimmune IgYwas unable to neutralize the hemagglutination ability of toxin A.

[0355] e) Assay of In Vitro Toxin A Neutralizing Activity

[0356] The ability of the anti-recombinant toxin A IgY (immune IgYantibodies raised against pMA1870-2680, the soluble recombinant bindingdomain protein expressed in pMAL, designated as Anti-tox. A-2 in FIG.14, and referred to as recombinant region 6) and pre-immune IgY,prepared as described in Example 8(c) above, to neutralize the cytotoxicactivity of toxin A was assessed in vitro using the CHO cellcytotoxicity assay, and toxin A (Tech Lab) at a concentration of 0.1μg/ml, as described in Example 8(d) above. As additional controls, theanti-native toxin A IgY (CTA) and pre-immune IgY preparations describedin Example 8(c) above were also tested. The results are shown in FIG.14.

[0357] The anti-recombinant toxin A IgY demonstrated only partialneutralization of the cytotoxic activity of toxin A, while thepre-immune IgY did not demonstrate any significant neutralizingactivity.

Example 14 In Vivo Neutralization of C. difficile Toxin A

[0358] The ability of avian antibodies (IgY) raised against recombinanttoxin A binding domain to neutralize the enterotoxin activity of C.difficile toxin A was evaluated in vivo using Golden Syrian hamsters.The Example involved:

[0359] (a) preparation of the avian anti-recombinant toxin A IgY fororal administration;

[0360] (b) in vivo protection of hamsters from C. difficile toxin Aenterotoxicity by treatment of toxin A with avian anti-recombinant toxinA IgY; and (c) histologic evaluation of hamster ceca.

[0361] a) Preparation of the Avian Anti-Recombinant Toxin A IgY for OralAdministration

[0362] Eggs were collected from hens which had been immunized with therecombinant C. difficile toxin A fragment pMA1870-2680 (described inExample 13, above). A second group of eggs purchased at a localsupermarket was used as a pre-immune (negative) control. Egg yolkimmunoglobulin (IgY) was extracted by PEG from the two groups of eggs asdescribed in Example 8(c), and the final IgY pellets were solubilized inone-fourth the original yolk volume using 0.1M carbonate buffer (mixtureof NaHCO₃ and N₂CO₃), pH 9.5. The basic carbonate buffer was used inorder to protect the toxin A from the acidic pH of the stomachenvironment.

[0363] b) In Vivo Protection of Hamsters Against C. difficile Toxin AEnterotoxicity by Treatment of Toxin A with Avian Anti-Recombinant ToxinA IgY

[0364] In order to assess the ability of the avian anti-recombinanttoxin A IgY, prepared in section (a) above to neutralize the in vivoenterotoxin activity of toxin A, an in vivo toxin neutralization modelwas developed using Golden Syrian hamsters. This model was based onpublished values for the minimum amount of toxin A required to elicitdiarrhea (0.08 mg toxin A/Kg body wt.) and death (0.16 mg toxin A/Kgbody wt.) in hamsters when administered orally (Lyerly et al. Infect.Immun., 47:349-352 (1985).

[0365] For the study, four separate experimental groups were used, witheach group consisting of 7 female Golden Syrian hamsters (CharlesRiver), approx. three and one-half weeks old, weighing approx. 50 gmseach. The animals were housed as groups of 3 and 4, and were offeredfood and water ad libitum through the entire length of the study.

[0366] For each animal, a mixture containing either 10 μg of toxin A(0.2 mg/Kg) or 30 μg of toxin A (0.6 mg/Kg) (C. difficile toxin A wasobtained from Tech Lab and 1 ml of either the anti-recombinant toxin AIgY or pre-immune IgY (from section (a) above) was prepared. Thesemixtures were incubated at 37° C. for 60 min. and were then administeredto the animals by the oral route. The animals were then observed for theonset of diarrhea and death for a period of 24 hrs. following theadministration of the toxin A+IgY mixtures, at the end of which time,the following results were tabulated and shown in Table 17: TABLE 17Study Outcome At 24 Hours Study Outcome at 24 Hours Experimental GroupHealthy¹ Diarrhea² Dead³ 10 μg Toxin A + Antitoxin 7 0 0 AgainstInterval 6 30 μg Toxin A + Antitoxin 7 0 0 Against Interval 6 10 μgToxin A + Pre-Immune 0 5 2 Serum 30 μg Toxin A + Pre-Immune 0 5 2

[0367] Pretreatment of toxin A at both doses tested, using theanti-recombinant toxin A IgY, prevented all overt symptoms of disease inhamsters. Therefore, pretreatment of C. difficile toxin A, using theanti-recombinant toxin A IgY, neutralized the in vivo enterotoxinactivity of the toxin A. In contrast, all animals from the two groupswhich received toxin A which had been pretreated using pre-immune IgYdeveloped disease symptoms which ranged from diarrhea to death. Thediarrhea which developed in the 5 animals which did not die in each ofthe two pre-immune groups, spontaneously resolved by the end of the 24hr. study period.

[0368] c) Histologic Evaluation of Hamster Ceca

[0369] In order to further assess the ability of anti-recombinant toxinA IgY to protect hamsters from the enterotoxin activity of toxin A,histologic evaluations were performed on the ceca of hamsters from thestudy described in section (b) above.

[0370] Three groups of animals were sacrificed in order to preparehistological specimens. The first group consisted of a singlerepresentative animal taken from each of the 4 groups of survivinghamsters at the conclusion of the study described in section (b) above.These animals represented the 24 hr. timepoint of the study.

[0371] The second group consisted of two animals which were not part ofthe study described above, but were separately treated with the sametoxin A+pre-immune IgY mixtures as described for the animals in section(b) above. Both of these hamsters developed diarrhea, and weresacrificed 8 hrs. after the time of administration of the toxinA+pre-immune IgY mixtures. At the time of sacrifice, both animals werepresenting symptoms of diarrhea. These animals represented the acutephase of the study.

[0372] The final group consisted of a single untreated hamster from thesame shipment of animals as those used for the two previous groups. Thisanimal served as the normal control.

[0373] Samples of cecal tissue were removed from the 7 animals describedabove, and were fixed overnight at 4° C. using 10% buffered formalin.The fixed tissues were paraffin-embedded, sectioned, and mounted onglass microscope slides. The tissue sections were then stained usinghematoxylin and eosin (H and E stain), and were examined by lightmicroscopy.

[0374] The tissues obtained from the two 24 hr. animals which receivedmixtures containing either 10 μg or 30 μg of toxin A andanti-recombinant toxin A IgY were indistinguishable from the normalcontrol, both in terms of gross pathology, as well as at the microscopiclevel. These observations provide further evidence for the ability ofanti-recombinant toxin A IgY to effectively neutralize the in vivoenterotoxin activity of C. difficile toxin A, and thus its ability toprevent acute or lasting toxin A-induced pathology.

[0375] In contrast, the tissues from the two 24 hr. animals whichreceived the toxin A+pre-immune IgY mixtures demonstrated significantpathology. In both of these groups, the mucosal layer was observed to beless organized than in the normal control tissue. The cytoplasm of theepithelial cells had a vacuolated appearance, and gaps were presentbetween the epithelium and the underlying cell layers. The laminapropria was largely absent. Intestinal villi and crypts weresignificantly diminished, and appeared to have been overgrown by aplanar layer of epithelial cells and fibroblasts. Therefore, althoughthese animals overtly appeared to recover from the acute symptoms oftoxin A intoxication, lasting pathologic alterations to the cecal mucosahad occurred.

[0376] The tissues obtained from the two acute animals which receivedmixtures of toxin A and pre-immune IgY demonstrated the most significantpathology. At the gross pathological level, both animals were observedto have severely distended ceca which were filled with watery,diarrhea-like material. At the microscopic level, the animal that wasgiven the mixture containing 10 μg of toxin A and pre-immune IgY wasfound to have a mucosal layer which had a ragged, damaged appearance,and a disorganized, compacted quality. The crypts were largely absent,and numerous breaks in the epithelium had occurred. There was also aninflux of erythrocytes into spaces between the epithelial layer and theunderlying tissue. The animal which had received the mixture containing30 μg of toxin A and pre-immune IgY demonstrated the most severepathology. The cecal tissue of this animal had an appearance verysimilar to that observed in animals which had died from C. difficiledisease. Widespread destruction of the mucosa was noted, and theepithelial layer had sloughed. Hemorrhagic areas containing largenumbers of erythrocytes were very prevalent. All semblance of normaltissue architecture was absent from this specimen. In terms of thepresentation of pathologic events, this in vivo hamster model of toxinA-intoxication correlates very closely with the pathologic consequencesof C. difficile disease in hamsters. The results presented in thisExample demonstrate that while anti-recombinant toxin A (Interval 6) IgYis capable of only partially neutralizing the cytotoxic activity of C.difficile toxin A, the same antibody effectively neutralizes 100% of thein vivo enterotoxin activity of the toxin. While it is not intended thatthis invention be limited to this mechanism, this may be due to thecytotoxicity and enterotoxicity of C. difficile Toxin A as two separateand distinct biological functions.

Example 15 In Vivo Neutralization of C Difficile Toxin A by AntibodiesAgainst Recombinant Toxin A Polypeptides

[0377] The ability of avian antibodies directed against the recombinantC. difficile toxin A fragment 1870-2680 (as expressed by pMA1870-2680;see Example 13) to neutralize the enterotoxic activity of toxin A wasdemonstrated in Example 14. The ability of avian antibodies (IgYs)directed against other recombinant toxin A epitopes to neutralize nativetoxin A in vivo was next evaluated. This example involved: (a) thepreparation of IgYs against recombinant toxin A polypeptides; (b) invivo protection of hamsters against toxin A by treatment withanti-recombinant toxin A IgYs and (c) quantification of specificantibody concentration in CTA and Interval 6 IgY PEG preparations.

[0378] The nucleotide sequence of the coding region of the entire toxinA protein is listed in SEQ ID NO:5. The amino acid sequence of theentire toxin A protein is listed in SEQ ID NO:6. The amino acid sequenceconsisting of amino acid residues 1870 through 2680 of toxin A is listedin SEQ ID NO:7. The amino acid sequence consisting of amino acidresidues 1870 through 1960 of toxin A is listed in SEQ ID NO:8.

[0379] a) Preparation of IgY's Against Recombinant Toxin A Polypeptides

[0380] Eggs were collected from Leghorn hens which have been immunizedwith recombinant C. difficile toxin A polypeptide fragments encompassingthe entire toxin A protein. The polypeptide fragments used as immunogenswere: 1) pMA 1870-2680 (Interval 6), 2) pPA1100-1450 (Interval 4), and3) a mixture of fragments consisting of pMA 30-300 (Interval 1), pMA300-660 (Interval 2), pMA 660-1100 (Interval 3) and pMA 1450-1870(Interval 5). This mixture of immunogens is referred to as Interval1235. The location of each interval within the toxin A molecule is shownin FIG. 15A. In FIG. 15A, the following abbreviations are used: pPrefers to the pET23 vector (New England BioLabs); pM refers to thepMAL™-c vector (New England BioLabs); A refers to toxin A; the numbersrefer to the amino acid interval expressed in the clone. (For example,the designation pMA30-300 indicates that the recombinant clone encodesamino acids 30-300 of toxin A and the vector used was pMAL™-c).

[0381] The recombinant proteins were generated as described in Example11. The IgYs were extracted and solubilized in 0.1M carbonate buffer pH9.5 for oral administration as described in Example 14(a). The IgYreactivities against each individual recombinant interval was evaluatedby ELISA as described in Example 13(c).

[0382] b) In Vivo Protection of Hamsters Against Toxin A by Treatmentwith Anti-Recombinant Toxin A Antibodies

[0383] The ability of antibodies raised against recombinant toxin Apolypeptides to provide in vivo protection against the enterotoxicactivity of toxin A was examined in the hamster model system. This assaywas performed as described in Example 14(b). Briefly, for each 40-50gram female Golden Syrian hamster (Charles River), 1 ml of IgY 4× (i.e.,resuspended in ¼ of the original yolk volume) PEG prep against Interval6, Interval 4 or Interval 1235 was mixed with 30 μg (LD₁₀₀ oral dose) ofC. difficile toxin A (Tech Lab). Preimmune IgY mixed with toxin A servedas a negative control. Antibodies raised against C. difficile toxoid A(Example 8) mixed with toxin A (CTA) served as a positive control. Themixture was incubated for 1 hour at 37° C. then orally administered tolightly etherized hamsters using an 18G feeding needle. The animals werethen observed for the onset of diarrhea and death for a period ofapproximately 24 hours. The results are shown in Table 18. TABLE 18Study Outcome After 24 Hours Treatment group Healthy¹ Diarrhea² Dead³Preimmune 0 0 7 CTA 5 0 0 Interval 6 6 1 0 Interval 4 0 1 6 Interval1235 0 0 7

[0384] Pre-treatment of toxin A with IgYs against Interval 6 preventeddiarrhea in 6 of 7 hamsters and completely prevented death in all 7. Incontrast, as with preimmune IgY, IgYs against Interval 4 and Interval1235 had no effect on the onset of diarrhea and death in the hamsters.

[0385] c) Quantification of Specific Antibody Concentration in CTA andInterval 6 IgY PEG Preparations

[0386] To determine the purity of IgY PEG preparations, an aliquot of apMA1870-2680 (Interval 6) IgY PEG preparation was chromatographed usingHPLC and a KW-803 sizing column (Shodex). The resulting profile ofabsorbance at 280 nm is shown in FIG. 16. The single large peakcorresponds to the predicted MW of IgY. Integration of the area underthe single large peak showed that greater than 95% of the protein elutedfrom the column was present in this single peak. This resultdemonstrated that the majority (>95%) of the material absorbing at 280nm in the PEG preparation corresponds to IgY. Therefore, absorbance at280 mm can be used to determine the total antibody concentration in PEGpreparations.

[0387] To determine the concentration of Interval 6-specific antibodies(expressed as percent of total antibody) within the CTA and pMA1870-2680(Interval 6) PEG preparations, defined quantities of these antibodypreparations were affinity purified on a pPA1870-2680(H) (shownschematically in FIG. 15B) affinity column and the specific antibodieswere quantified. In FIG. 15B the following abbreviations are used: pPrefers to the pET23 vector (New England BioLabs); pM refers to thepMAL™-c vector (New England BioLabs); pG refers to the pGEX vector(Pharmacia); pB refers to the PinPoint™ Xa vector (Promega); A refers totoxin A; the numbers refer to the amino acid interval expressed in theclone. The solid black ovals represent the MBP; the hatched ovalsrepresent glutathione S-transferase; the hatched circles represent thebiotin tag; and HHH represents the poly-histidine tag.

[0388] An affinity column containing recombinant toxin A repeat proteinwas made as follows. Four ml of PBS-washed Actigel resin (Sterogene) wascoupled with 5-10 mg of pPA1870-2680 inclusion body protein [prepared asdescribed in Example (17) and dialyzed into PBS] in a 15 ml tube(Falcon) containing {fraction (1/10)} final volume Ald-coupling solution(1 M sodium cyanoborohydride). Aliquots of the supernatant from thecoupling reactions, before and after coupling, were assessed byCoomassie staining of 7.5% SDS-PAGE gels. Based upon protein bandintensities, greater than 6 mg of recombinant protein was coupled to theresin. The resin was poured into a 10 ml column (BioRad), washedextensively with PBS, pre-eluted with 4 M guanidine-HCl (in 10 mMTris-HCl, pH 8.0; 0.005% thimerosal) and re-equilibrated with PBS. Thecolumn was stored at 4° C.

[0389] Aliquots of a pMA1870-2680 (Interval 6) or a CTA IgY polyclonalantibody preparation (PEG prep) were affinity purified on the aboveaffinity column as follows. The column was attached to an UV monitor(ISCO) and washed with PBS. For pMA1870-2680 IgY purification, a 2×PEGprep (filter sterilized using a 0.45μ filter; approximately 500 mg totalIgY) was applied. The column was washed with PBS until the baseline wasre-established (the column flow-through was saved), washed with BBSTweento elute nonspecifically binding antibodies and re-equilibrated withPBS. Bound antibody was eluted from the column in 4 M guanidine-HCl (in10 mM Tris-HCl, pH 8.0; 0.005% thimerosal). The entire elution peak wascollected in a 15 ml tube (Falcon). The column was re-equilibrated andthe column eluate was re-chromatographed as described above. Theantibody preparation was quantified by UV absorbance (the elution bufferwas used to zero the spectrophotometer). Total purified antibody wasapproximately 9 mg and 1 mg from the first and second chromatographypasses, respectively. The low yield from the second pass indicated thatmost specific antibodies were removed by the first round ofchromatography. The estimated percentage of Interval 6 specificantibodies in the pMA1870-2680 PEG prep is approximately 2%.

[0390] The percentage of Interval 6 specific antibodies in the CTA PEGprep was determined (utilizing the same column and methodology describedabove) to be approximately 0.5% of total IgY.

[0391] A 4×PEG prep contains approximately 20 mg/ml IgY. Thus in b)above, approximately 400 μg specific antibody in the Interval 6 PEG prepneutralized 30 μg toxin A in vivo.

Example 16 In Vivo Treatment of C. difficile Disease in Hamsters byRecombinant Interval 6 Antibodies

[0392] The ability of antibodies directed against recombinant Interval 6of toxin A to protect hamsters in vivo from C. difficile disease wasexamined. This example involved: (a) prophylactic treatment of C.difficile disease and (b) therapeutic treatment of C. difficile disease.

[0393] a) Prophylactic Treatment of C. difficile Disease

[0394] This experiment was performed as described in Example 9(b). Threegroups each consisting of 7 female 100 gram Syrian hamsters (CharlesRiver) were prophylactically treated with either preimmune IgYs, IgYsagainst native toxin A and B [CTAB; see Example 8 (a) and (b)] or IgYsagainst Interval 6. IgYs were prepared as 4×PEG preparations asdescribed in Example 9(a).

[0395] The animals were orally dosed 3 times daily, roughly at 4 hourintervals, for 12 days with 1 ml antibody preparations diluted inEnsure®. Using estimates of specific antibody concentration from Example15(c), each dose of the Interval 6 antibody prep contained approximately400 μg of specific antibody. On day 2 each hamster was predisposed to C.difficile infection by the oral administration of 3.0 mg ofClindamycin-HCl (Sigma) in 1 ml of water. On day 3 the hamsters wereorally challenged with 1 ml of C. difficile inoculum strain ATCC 43596in sterile saline containing approximately 100 organisms. The animalswere then observed for the onset of diarrhea and subsequent death duringthe treatment period. The results are shown in Table 19. TABLE 19Lethality After 12 Days Of Treatment Number Number Treatment AnimalsAnimals Group Alive Dead Preimmune 0 7 CTAB 6 1 Interval 6 7 0

[0396] Treatment of hamsters with orally-administered IgYs againstInterval 6 successfully protected 7 out of 7 (100%) of the animals fromC. difficile disease. One of the hamsters in this group presented withdiarrhea which subsequently resolved during the course of treatment. Asshown previously in Example 9, antibodies to native toxin A and toxin Bwere highly protective. In this Example, 6 out of 7 animals survived inthe CTAB treatment group. All of the hamsters treated with preimmunesera came down with diarrhea and died. The survivors in both the CTABand Interval 6 groups remained healthy throughout a 12 daypost-treatment period. In particular, 6 out of 7 Interval 6-treatedhamsters survived at least 2 weeks after termination of treatment whichsuggests that these antibodies provide a long-lasting cure. Theseresults represent the first demonstration that antibodies generatedagainst a recombinant region of toxin A can prevent CDAD whenadministered passively to animals. These results also indicate thatantibodies raised against Interval 6 alone may be sufficient to protectanimals from C. difficile disease when administered prophylactically.

[0397] Previously others had raised antibodies against toxin A byactively immunizing hamsters against a recombinant polypeptide locatedwithin the Interval 6 region [Lyerly, D. M., et al. (1990) Curr.Microbiol. 21:29]. FIG. 17 shows schematically the location of theLyerly, et al. intra-Interval 6 recombinant protein (cloned into the pUCvector) in comparison with the complete Interval 6 construct(pMA1870-2680) used herein to generate neutralizing antibodies directedagainst toxin A. In FIG. 17, the solid black oval represents the MBPwhich is fused to the toxin A Interval 6 in pMA1870-2680.

[0398] The Lyerly, et al. antibodies (intra-Interval 6) were only ableto partially protect hamsters against C. difficile infection in terms ofsurvival (4 out of 8 animals survived) and furthermore, these antibodiesdid not prevent diarrhea in any of the animals. Additionally, animalstreated with the intra-Interval 6 antibodies [Lyerly, et al. (1990),supra] died when treatment was removed.

[0399] In contrast, the experiment shown above demonstrates that passiveadministration of anti-Interval 6 antibodies prevented diarrhea in 6 outof 7 animals and completely prevented death due to CDAD. Furthermore, asdiscussed above, passive administration of the anti-Interval 6antibodies provides a long lasting cure (i.e., treatment could bewithdrawn without incident).

[0400] b) Therapeutic Treatment of C. difficile Disease: In VivoTreatment of an Established C. difficile Infection in Hamsters withRecombinant Interval 6 Antibodies

[0401] The ability of antibodies against recombinant interval 6 of toxinA to therapeutically treat C. difficile disease was examined. Theexperiment was performed essentially as described in Example 10(b).Three groups, each containing seven to eight female Golden Syrianhamsters (100 g each; Charles River) were treated with either preimmuneIgY, IgYs against native toxin A and toxin B (CTAB) and IgYs againstInterval 6. The antibodies were prepared as described above as 4×PEGpreparations.

[0402] The hamsters were first predisposed to C. difficile infectionwith a 3 mg dose of Clindamycin-HCl (Sigma) administered orally in 1 mlof water. Approximately 24 hrs later, the animals were orally challengedwith 1 ml of C. difficile strain ATCC 43596 in sterile saline containingapproximately 200 organisms. One day after infection, the presence oftoxin A and B was determined in the feces of the hamsters using acommercial immunoassay kit (Cytoclone A+B EPA, Cambridge Biotech) toverify establishment of infection. Four members of each group wererandomly selected and tested. Feces from an uninfected hamster wastested as a negative control. All infected animals tested positive forthe presence of toxin according to the manufacturer's procedure. Theinitiation of treatment then started approximately 24 hr post-infection.

[0403] The animals were dosed daily at roughly 4 hr intervals with 1 mlantibody preparation diluted in Ensure® (Ross Labs). The amount ofspecific antibodies given per dose (determined by affinity purification)was estimated to be about 400 μg of anti-Interval 6 IgY (for animals inthe Interval 6 group) and 100 μg and 70 μg of anti-toxin A (Interval6-specific) and anti-toxin B (Interval 3-specific; see Example 19),respectively, for the CTAB preparation. The animals were treated for 9days and then observed for an additional 4 days for the presence ofdiarrhea and death. The results indicating the number of survivors andthe number of dead 4 days post-infection are shown in Table 20. TABLE 20In vivo Therapeutic Treatment With Interval 6 Antibodies Number NumberTreatment Animals Animals Group Alive Dead Preimmune 4 3 CTAB 8 0Interval 6 8 0

[0404] Antibodies directed against both Interval 6 and CTAB successfullyprevented death from C. difficile when therapeutically administered 24hr after infection. This result is significant since many investigatorsbegin therapeutic treatment of hamsters with existing drugs (e.g.,vancomycin, phenelfamycins, tiacumicins, etc.) 8 hr post-infection[Swanson, et al. (1991) Antimicrobial Agents and Chemotherapy 35:1108and (1989) J. Antibiotics 42:94].

[0405] Forty-two percent of hamsters treated with preimmune IgY diedfrom CDAD. While the anti-Interval 6 antibodies prevented death in thetreated hamsters, they did not eliminate all symptoms of CDAD as 3animals presented with slight diarrhea. In addition, one CTAB-treatedand one preimmune-treated animal also had diarrhea 14 dayspost-infection. These results indicate that anti-Interval 6 antibodiesprovide an effective means of therapy for CDAD.

Example 17 Induction of Toxin A Neutralizing Antibodies Requires SolubleInterval 6 Protein

[0406] As shown in Examples 11(d) and 15, expression of recombinantproteins in E coli may result in the production of either soluble orinsoluble protein. If insoluble protein is produced, the recombinantprotein is solubilized prior to immunization of animals. To determinewhether, one or both of the soluble or insoluble recombinant proteinscould be used to generate neutralizing antibodies to toxin A, thefollowing experiment was performed. This example involved a) expressionof the toxin A repeats and subfragments of these repeats in E. coliusing a variety of expression vectors; b) identification of recombinanttoxin A repeats and sub-regions to which neutralizing antibodies bind;and c) determination of the neutralization ability of antibodies raisedagainst soluble and insoluble toxin A repeat immunogen.

[0407] a) Expression of the Toxin A Repeats and Subfragments of TheseRepeats in E. coli Using a Variety of Expression Vectors

[0408] The Interval 6 immunogen utilized in Examples 15 and 16 was thepMA1870-2680 protein, in which the toxin A repeats are expressed as asoluble fusion protein with the MBP (described in Example 11).Interestingly, expression of this region (from the SpeI site to the endof the repeats, see FIG. 15B) in three other expression constructs, aseither native (pPA1870-2680), poly-His tagged [pPA1870-2680 (H)] orbiotin-tagged (pBA1870-2680) proteins resulted in completely insolubleprotein upon induction of the bacterial host (see FIG. 15B). The hoststrain BL21 (Novagen) was used for expression of pBA1870-2680 and hoststrain BL21(DE3) (Novagen) was used for expression of pPA1870-2680 andpPA1870-2680(H). These insoluble proteins accumulated to high levels ininclusion bodies. Expression of recombinant plasmids in E. coli hostcells grown in 2×YT medium was performed as described [Williams, et al.(1995), supra].

[0409] As summarized in FIG. 15B, expression of fragments of the toxin Arepeats (as either N-terminal SpeI-EcoRI fragments, or C-terminalEcoRI-end fragments) also yielded high levels of insoluble protein usingpGEX (pGA1870-2190), PinPoint™-Xa (pBA1870-2190 and pBA2250-2680) andpET expression systems (pPA1870-2190). The pGEX and pET expressionsystems are described in Example 11. The PinPoint™-Xa expression systemdrives the expression of fusion proteins in E. coli. Fusion proteinsfrom PinPoint™-Xa vectors contain a biotin tag at the amino-terminal endand can be affinity purified SoftLink™ Soft Release avidin resin(Promega) under mild denaturing conditions (5 mM biotin).

[0410] The solubility of expressed proteins from the pPG1870-2190 andpPA1870-2190 expression constructs was determined after induction ofrecombinant protein expression under conditions reported to enhanceprotein solubility [These conditions comprise growth of the host atreduced temperature (30° C.) and the utilization of high (1 mM IPTG) orlow (0.1 mM IPTG) concentrations of inducer [Williams et al. (1995),supra]. All expressed recombinant toxin A protein was insoluble underthese conditions. Thus, expression of these fragments of the toxin Arepeats in pET and pGEX expression vectors results in the production ofinsoluble recombinant protein even when the host cells are grown atreduced temperature and using lower concentrations of the inducer.Although expression of these fragments in pMal vectors yielded affinitypurifiable soluble fusion protein, the protein was either predominantlyinsoluble (pMA1870-2190) or unstable (pMA2250-2650). Attempts tosolubilize expressed protein from the pMA1870-2190 expression constructusing reduced temperature or lower inducer concentration (as describedabove) did not improve fusion protein solubility.

[0411] Collectively, these results demonstrate that expression of thetoxin A repeat region in E. coli results in the production of insolublerecombinant protein, when expressed as either large (aa 1870-2680) orsmall (aa 1870-2190 or aa 2250-2680) fragments, in a variety ofexpression vectors (native or poly-his tagged pET, pGEX or PinPoint™-Xavectors), utilizing growth conditions shown to enhance proteinsolubility. The exception to this rule were fusions with the MBP, whichenhanced protein solubility, either partially (pMA1870-2190) or fully(pMA1870-2680).

[0412] b) Identification of Recombinant Toxin A Repeats and Sub-Regionsto Which Neutralizing Antibodies Bind

[0413] Toxin A repeat regions to which neutralizing antibodies bind wereidentified by utilizing recombinant toxin A repeat region proteinsexpressed as soluble or insoluble proteins to deplete protectiveantibodies from a polyclonal pool of antibodies against native C.difficile toxin A. An in vivo assay was developed to evaluate proteinsfor the ability to bind neutralizing antibodies.

[0414] The rational for this assay is as follows. Recombinant proteinswere first pre-mixed with antibodies against native toxin A (CTAantibody; generated in Example 8) and allowed to react. Subsequently, C.difficile toxin A was added at a concentration lethal to hamsters andthe mixture was administered to hamsters via IP injection. If therecombinant protein contains neutralizing epitopes, the CTA antibodieswould lose their ability to bind toxin A resulting in diarrhea and/ordeath of the hamsters.

[0415] The assay was performed as follows. The lethal dose of toxin Awhen delivered orally to nine 40 to 50 g Golden Syrian hamsters (Sasco)was determined to be 10 to 30 μg. The PEG-purified CTA antibodypreparation was diluted to 0.5× concentration (i.e., the antibodies werediluted at twice the original yolk volume) in 0.1 M carbonate buffer, pH9.5. The antibodies were diluted in carbonate buffer to protect themfrom acid degradation in the stomach. The concentration of 0.5× was usedbecause it was found to be the lowest effective concentration againsttoxin A. The concentration of Interval 6-specific antibodies in the0.5×CTA prep was estimated to be 10-15 μg/ml (estimated using the methoddescribed in Example 15).

[0416] The inclusion body preparation [insoluble Interval 6 protein;pPA1870-2680(H)] an d the soluble Interval 6 protein [α]1870-2680; seeFIG. 151 were both compared for their ability to bind to neutralizingantibodies against C. difficile toxin A (CTA). Specifically, 1 to 2 mgof recombinant protein was mixed with 5 ml of a 0.5×CTA antibody prep(estimated to contain 60-70 μg of Interval 6-specific antibody). Afterincubation for 1 hr at 37° C., CTA (Tech Lab) at a final concentrationof 30 μg/ml was added and incubated for another 1 hr at 37° C. One ml ofthis mixture containing 30 μg of toxin A (and 10-15 μg of Interval6-specific antibody) was administered orally to 40-50 g Golden Syrianhamsters (Sasco). Recombinant proteins that result in the loss ofneutralizing capacity of the CTA antibody would indicate that thoseproteins contain neutralizing epitopes. Preimmune and CTA antibodies(both at 0.5×) without the addition of any recombinant protein served asnegative and positive controls, respectively.

[0417] Two other inclusion body preparations, both expressed asinsoluble products in the pET vector, were tested; one containing adifferent insert (toxin B fragment) other than Interval 6 calledpPB1850-2070 (see FIG. 18) which serves as a control for insolubleInterval 6, the other was a truncated version of the Interval 6 regioncalled pPA1870-2190 (see FIG. 15B). The results of this experiment areshown in Table 21. TABLE 21 Binding Of Neutralizing Antibodies BySoluble Interval 6 Protein Study Outcome After 24 Hours Treatment Group¹Healthy² Diarrhea³ Dead⁴ Preimmune Ab 0 3 2 CTA Ab 4 1 0 CTA Ab + Int 6(soluble) 1 2 2 CTA Ab + Int 6 (insoluble) 5 0 0 CTA Ab + pPB1850-2070 50 0 CTA Ab + pPA1870-2190 5 0 0

[0418] Preimmune antibody was ineffective against toxin A, whileanti-CTA antibodies at a dilute 0.5× concentration almost completelyprotected the hamsters against the enterotoxic effects of CTA. Theaddition of recombinant proteins pPB1850-2070 or pPA1870-2190 to theanti-CTA antibody had no effect upon its protective ability, indicatingthat these recombinant proteins do not bind to neutralizing antibodies.On the other hand, recombinant Interval 6 protein was able to bind toneutralizing anti-CTA antibodies and neutralized the in vivo protectiveeffect of the anti-CTA antibodies. Four out of five animals in the grouptreated with anti-CTA antibodies mixed with soluble Interval 6 proteinexhibited toxin associated toxicity (diarrhea and death). Moreover, theresults showed that Interval 6 protein must be expressed as a solubleproduct in order for it to bind to neutralizing anti-CTA antibodiessince the addition of insoluble Interval 6 protein had no effect on theneutralizing capacity of the CTA antibody prep.

[0419] c) Determination of Neutralization Ability of Antibodies RaisedAgainst Soluble and Insoluble Toxin A Repeat Immunogen

[0420] To determine if neutralizing antibodies are induced againstsolubilized inclusion bodies, insoluble toxin A repeat protein wassolubilized and specific antibodies were raised in chickens. InsolublepPA1870-2680 protein was solubilized using the method described inWilliams et al. (1995), supra. Briefly, induced cultures (500 ml) werepelleted by centrifugation at 3,000×g for 10 min at 4° C. The cellpellets were resuspended thoroughly in 10 ml of inclusion bodysonication buffer (25 mM HEPES pH 7.7, 100 mM KCl, 12.5 mM MgCl₂, 20%glycerol, 0.1% (v/v) Nonidet P-40, 1 mM DTT). The suspension wastransferred to a 30 ml non-glass centrifuge tube. Five hundred μl of 10mg/ml lysozyme was added and the tubes were incubated on ice for 30 min.The suspension was then frozen at −70° C. for at least 1 hr. Thesuspension was thawed rapidly in a water bath at room temperature andthen placed on ice. The suspension was then sonicated using at leasteight 15 sec bursts of the microprobe (Branson Sonicator Model No. 450)with intermittent cooling on ice.

[0421] The sonicated suspension was transferred to a 35 ml Oakridge tubeand centrifuged at 6,000×g for 10 min at 4° C. to pellet the inclusionbodies. The pellet was washed 2 times by pipetting or vortexing infresh, ice-cold RIPA buffer [0.1% SDS, 1% Triton X-100, 1% sodiumdeoxycholate in TBS (25 mM Tris-Cl pH 7.5, 150 mM NaCl)]. The inclusionbodies were recentrifuged after each wash. The inclusion bodies weredried and transferred using a small metal spatula to a 15 ml tube(Falcon). One ml of 10% SDS was added and the pellet was solubilized bygently pipetting the solution up and down using a 1 ml micropipettor.The solubilization was facilitated by heating the sample to 95° C. whennecessary.

[0422] Once the inclusion bodies were in solution, the samples werediluted with 9 volumes of PBS. The protein solutions were dialyzedovernight against a 100-fold volume of PBS containing 0.05% SDS at roomtemperature. The dialysis buffer was then changed to PBS containing0.01% SDS and the samples were dialyzed for several hours to overnightat room temperature. The samples were stored at 4° C. until used. Priorto further use, the samples were warmed to room temperature to allow anyprecipitated SDS to go back into solution.

[0423] The inclusion body preparation was used to immunize hens. Theprotein was dialyzed into PBS and emulsified with approximately equalvolumes of CFA for the initial immunization or IFA for subsequentbooster immunizations. On day zero, for each of the recombinantrecombinant preparations, two egg laying white Leghorn hens were eachinjected at multiple sites (IM and SC) with 1 ml of recombinantprotein-adjuvant mixture containing approximately 0.5-1.5 mg ofrecombinant protein. Booster immunizations of 1.0 mg were given of days14 and day 28. Eggs were collected on day 32 and the antibody isolatedusing PEG as described in Example 14(a). High titers of toxin A specificantibodies were present (as assayed by ELISA, using the method describedin Example 13). Titers were determined for both antibodies againstrecombinant polypeptides pPA1870-2680 and pMA1870-2680 and were found tobe comparable at >1:62,500.

[0424] Antibodies against soluble Interval 6 (pMA1870-2680) andinsoluble Interval 6 [(inclusion body), pPA1870-2680] were tested forneutralizing ability against toxin A using the in vivo assay describedin Example 15(b). Preimmune antibodies and antibodies against toxin A(CTA) served as negative and positive controls, respectively. Theresults are shown in Table 22. TABLE 22 Neutralization Of Toxin A ByAntibodies Against Soluble Interval 6 Protein Study Outcome After 24Hours Antibody Treatment Group Healthy¹ Diarrhea² Dead³ Preimmune 1 0 4CTA 5 0 0 Interval 6 (Soluble)⁴ 5 0 0 Interval 6 (Insoluble) 0 2 3

[0425] Antibodies raised against native toxin A were protective whilepreimmune antibodies had little effect. As found using the in vitro CHOassay [described in Example 8(d)] where antibodies raised against thesoluble Interval 6 could partially neutralize the effects of toxin A,here they were able to completely neutralize toxin A in vivo. Incontrast, the antibodies raised against the insoluble Interval 6 wasunable to neutralize the effects of toxin A in vivo as shown above(Table 22) and in vitro as shown in the CHO assay [described in Example8(d)].

[0426] These results demonstrate that soluble toxin A repeat immunogenis necessary to induce the production of neutralizing antibodies inchickens, and that the generation of such soluble immunogen is obtainedonly with a specific expression vector (pMal) containing the toxin Aregion spanning aa 1870-2680. That is to say, insoluble protein that issubsequently solubilized does not result in a toxin A antigen that willelicit a neutralizing antibody.

Example 18 Cloning and Expression of the C. difficile Toxin B Gene

[0427] The toxin B gene has been cloned and sequenced; the amino acidsequence deduced from the cloned nucleotide sequence predicts a MW of269.7 kD for toxin B [Barroso et al., Nucl. Acids Res. 18:4004 (1990)].The nucleotide sequence of the coding region of the entire toxin B geneis listed in SEQ ID NO:9. The amino acid sequence of the entire toxin Bprotein is listed in SEQ ID NO:10. The amino acid sequence consisting ofamino acid residues 1850 through 2360 of toxin B is listed in SEQ IDNO:11. The amino acid sequence consisting of amino acid residues 1750through 2360 of toxin B is listed in SEQ ID NO:12.

[0428] Given the expense and difficulty of isolating native toxin Bprotein, it would be advantageous to use simple and inexpensiveprocaryotic expression systems to produce and purify high levels ofrecombinant toxin B protein for immunization purposes. Ideally, theisolated recombinant protein would be soluble in order to preservenative antigenicity, since solubilized inclusion body proteins often donot fold into native conformations. Indeed as shown in Example 17,neutralizing antibodies against recombinant toxin A were only obtainedwhen soluble recombinant toxin A polypeptides were used as theimmunogen. To allow ease of purification, the recombinant protein shouldbe expressed to levels greater than 1 mg/liter of E. coli culture.

[0429] To determine whether high levels of recombinant toxin B proteincould be produced in E. coli, fragments of the toxin B gene were clonedinto various prokaryotic expression vectors, and assessed for theability to express recombinant toxin B protein in E. coli. This Exampleinvolved (a) cloning of the toxin B gene and (b) expression of the toxinB gene in E. coli.

[0430] a) Cloning of the Toxin B Gene

[0431] The toxin B gene was cloned using PCR amplification from C.difficile genomic DNA. Initially, the gene was cloned in two overlappingfragments, using primer pairs P5/P6 and P7/P8. The location of theseprimers along the toxin B gene is shown schematically in FIG. 18. Thesequence of each of these primers is: P5: 5′ TAGAAAAAATGGCAAATGT 3′ (SEQID NO:11); P6: 5′ TTTCATCTTGTA GAGTCAAAG 3′ (SEQ ID NO:12); P7: 5′GATGCCACAAGATGATTTAGTG 3′ (SEQ ID NO:13); and P8: 5′CTAATTGAGCTGTATCAGGATC 3′ (SEQ ID NO:14).

[0432]FIG. 18 also shows the location of the following primers along thetoxin B gene: P9 which consists of the sequence 5′CGGAATTCCTAGAAAAAATGGCAA ATG 3′ (SEQ ID NO:15); P10 which consists ofthe sequence 5′ GCTCTAGAATGA CCATAAGCTAGCCA 3′ (SEQ ID NO:16); P11 whichconsists of the sequence 5′ CGGAATTCGAGTTGGTAGAAAGGTGGA 3′ (SEQ IDNO:17); P13 which consists of the sequence 5′CGGAATTCGGTTATTATCTTAAGGATG 3′ (SEQ ID NO:18); and P14 which consists ofthe sequence 5′ CGGAATTCTTGATAACTGGAT TTGTGAC 3′ (SEQ ID NO:19). Theamino acid sequence consisting of amino acid residues 1852 through 2362of toxin B is listed in SEQ ID NO:20. The amino acid sequence consistingof amino acid residues 1755 through 2362 of toxin B is listed in SEQ IDNO:21.

[0433]Clostridium difficile VPI strain 10463 was obtained from theAmerican Type Culture Collection (ATCC 43255) and grown under anaerobicconditions in brain-heart infusion medium (Becton Dickinson). Highmolecular-weight C. difficile DNA was isolated essentially as described[Wren and Tabaqchali (1987) J. Clin. Microbiol., 25:2402], except 1) 100μg/ml proteinase K in 0.5% SDS was used to disrupt the bacteria and 2)cetytrimethylammonium bromide (CTAB) precipitation [as described byAusubel et al., Eds., Current Protocols in Molecular Biology, Vol. 2(1989) Current Protocols] was used to remove carbohydrates from thecleared lysate. Briefly, after disruption of the bacteria withproteinase K and SDS, the solution is adjusted to approximately 0.7 MNaCl by the addition of a 1/7 volume of 5M NaCl. A 1/10 volume ofCTAB/NaCl (10% CTAB in 0.7 M NaCl) solution was added and the solutionwas mixed thoroughly and incubated 10 min at 65° C. An equal volume ofchloroform/isoamyl alcohol (24:1) was added and the phases werethoroughly mixed. The organic and aqueous phases were separated bycentrifugation in a microfuge for 5 min. The aqueous supernatant wasremoved and extracted with phenol/chloroform/isoamyl alcohol (25:24:1).The phases were separated by centrifugation in a microfuge for 5 min.The supernatant was transferred to a fresh tube and the DNA wasprecipitated with isopropanol. The DNA precipitate was pelleted by briefcentrifugation in a microfuge. The DNA pellet was washed with 70%ethanol to remove residual CTAB. The DNA pellet was then dried andredissolved in TE buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA). Theintegrity and yield of genomic DNA was assessed by comparison with aserial dilution of uncut lambda DNA after electrophoresis on an agarosegel.

[0434] Toxin B fragments were cloned by PCR utilizing a proofreadingthermostable DNA polymerase [native Pfu polymerase (Stratagene)]. Thehigh fidelity of this polymerase reduces the mutation problemsassociated with amplification by error prone polymerases (e.g., Taqpolymerase). PCR amplification was performed using the PCR primer pairsP5 (SEQ ID NO:11) with P6 (SEQ ID NO:12) and P7 (SEQ ID NO:13) with P8(SEQ ID NO:14) in 50 μl reactions containing 10 mM Tris-HCl pH8.3, 50 mMKCl, 1.5 mM MgCl₂, 200 μM of each dNTP, 0.2 μM each primer, and 50 ng C.difficile genomic DNA. Reactions were overlaid with 100 μl mineral oil,heated to 94° C. for 4 min, 0.5 μl native Pfu polymerase (Stratagene)was added, and the reactions were cycled 30 times at 94° C. for 1 min,50° C. for 1 min, 72° C. (2 min for each kb of sequence to beamplified), followed by 10 min at 72° C. Duplicate reactions werepooled, chloroform extracted, and ethanol precipitated. After washing in70% ethanol, the pellets were resuspended in 50 μl TE buffer (10 mMTris-HCl pH8.0, 1 mM EDTA).

[0435] The P5/P6 amplification product was cloned into pUC19 as outlinedbelow. 10 μl aliquots of DNA were gel purified using the Prep-a-Gene kit(BioRad), and ligated to SmaI restricted pUC19 vector. Recombinantclones were isolated and confirmed by restriction digestion usingstandard recombinant molecular biology techniques (Sambrook et al.,1989). Inserts from two independent isolates were identified in whichthe toxin B insert was oriented such that the vector. BamHI and SacIsites were 5′ and 3′ oriented, respectively (pUCB10-1530). Theinsert-containing BamHI/SacI fragment was cloned into similarly cutpET23a-c vector DNA, and protein expression was induced in small scalecultures (5 ml) of identified clones.

[0436] Total protein extracts were isolated, resolved on SDS-PAGE gels,and toxin B protein identified by Western analysis utilizing a goatanti-toxin B affinity purified antibody (Tech Lab). Procedures forprotein induction, SDS-PAGE, and Western blot analysis were performed asdescribed in Williams et al. (1995), supra. In brief, 5 ml cultures ofbacteria grown in 2XYT containing 100 μg/ml ampicillin containing theappropriate recombinant clone were induced to express recombinantprotein by addition of IPTG to 1 mM. The E. coli hosts used were:BL21(DE3) or BL21(DE3)LysS (Novagen) for pET plasmids.

[0437] Cultures were induced by the addition of IPTG to a finalconcentration of 1.0 mM when the cell density reached 0.5 OD₆₀₀, andinduced protein was allowed to accumulate for two hrs after induction.Protein samples were prepared by pelleting 1 ml aliquots of bacteria bycentrifugation (1 min in microfuge), and resuspension of the pelletedbacteria in 150 μl of 2×SDS-PAGE sample buffer (0.125 mM Tris-HCl pH6.8, 2 mM EDTA, 6% SDS, 20% glycerol, 0.025% bromophenol blue;β-mercaptoethanol is added to 5% before use). The samples were heated to95° C. for 5 min, then cooled and 5 or 10 μls loaded on 7.5% SDS-PAGEgels. High molecular weight protein markers (BioRad) were also loaded,to allow estimation of the MW of identified fusion proteins. Afterelectrophoresis, protein was detected either generally by staining thegels with Coomassie Blue, or specifically, by blotting to nitrocellulosefor Western blot detection of specific immunoreactive protein. The MW ofinduced toxin B reactive protein allowed the integrity of the toxin Breading frame to be determined.

[0438] The pET23b recombinant (pPB10-1530) expressed high MW recombinanttoxin B reactive protein, consistent with predicted values. Thisconfirmed that frame terminating errors had not occurred during PCRamplification. A pET23b expression clone containing the 10-1750aainterval of the toxin B gene was constructed, by fusion of theEcoRV-SpeI fragment of the P7/P8 amplification product to the P5-EcoRVinterval of the P5/P6 amplification product (see FIG. 18) in pPB10-1530.The integrity of this clone (pPB10-1750) was confirmed by restrictionmapping, and Western blot detection of expressed recombinant toxin Bprotein. Levels of induced protein from both pPB10-1530 and pPB10-1750were too low to facilitate purification of usable amounts of recombinanttoxin B protein. The remaining 1750-2360 aa interval was directly clonedinto pMAL or pET expression vectors from the P7/P8 amplification productas described below.

[0439] b) Expression of the Toxin B Gene

[0440] i) Overview of Expression Methodologies

[0441] Fragments of the toxin B gene were expressed as either native orfusion proteins in E. coli. Native proteins were expressed in either thepET23a-c or pET16b expression vectors (Novagen). The pET23 vectorscontain an extensive polylinker sequence in all three reading frames(a-c vectors) followed by a C-terminal poly-histidine repeat. The pET16bvector contains a N-terminal poly-histidine sequence immediately 5′ to asmall polylinker. The poly-histidine sequence binds to Ni-Chelatecolumns and allows affinity purification of tagged target proteins[Williams et al. (1995), supra]. These affinity tags are small (10 aafor pET16b, 6 aa for pET23) allowing the expression and affinitypurification of native proteins with only limited amounts of foreignsequences.

[0442] An N-terminal histidine-tagged derivative of pET16b containing anextensive cloning cassette was constructed to facilitate cloning ofN-terminal poly-histidine tagged toxin B expressing constructs. This wasaccomplished by replacement of the promoter region of the pET23a and bvectors with that of the pET16b expression vector. Each vector wasrestricted with BglII and NdeI, and the reactions resolved on a 1.2%agarose gel. The pET16b promoter region (contained in a 200 bpBglII-NdeI fragment) and the promoter-less pET23 a or b vectors were cutfrom the gel, mixed and Prep-A-Gene (BioRad) purified. The eluted DNAwas ligated, and transformants screened for promoter replacement by NcoIdigestion of purified plasmid DNA (the pET16b promoter contains thissite, the pET23 promoter does not). These clones (denoted pETHisa or b)contain the pET16b promoter (consisting of a pT7-lac promoter, ribosomebinding site and poly-histidine (10aa) sequence) fused at the NdeI siteto the extensive pET23 polylinker.

[0443] All MBP fusion proteins were constructed and expressed in thepMAL™-c or pMAL™-p2 vectors (New England Biolabs) in which the proteinof interest is expressed as a C-terminal fusion with MBP. All pETplasmids were expressed in either the BL21(DE3) or BL21(DE3)LysSexpression hosts, while pMal plasmids were expressed in the BL21 host.

[0444] Large scale (500 mls to 1 liter) cultures of each recombinantwere grown in 2×YT broth, induced, and soluble protein fractions wereisolated as described [Williams, et al. (1995), supra]. The solubleprotein extracts were affinity chromatographed to isolate recombinantfusion protein, as described [Williams et al., (1995) supra]. In brief,extracts containing tagged pET fusions were chromatographed on a nickelchelate column, and eluted using imidazole salts or low pH (pH 4.0) asdescribed by the distributor (Novagen or Qiagen). Extracts containingsoluble pMAL fusion protein were prepared and chromatographed in PBSbuffer over an amylose resin (New England Biolabs) column, and elutedwith PBS containing 10 mM maltose as described [Williams et al. (1995),supra].

[0445] ii) Overview of Toxin B Expression

[0446] In both large expression constructs described in (a) above, onlylow level (i.e., less than 1 mg/liter of intact or nondegradedrecombinant protein) expression of recombinant protein was detected. Anumber of expression constructs containing smaller fragments of thetoxin B gene were then constructed, to determine if small regions of thegene can be expressed to high levels (i.e., greater than 1 mg/literintact protein) without extensive protein degradation. All wereconstructed by in frame fusions of convenient toxin B restrictionfragments to either the pMAL-c, pET23a-c, pET 16b or pETHisa-bexpression vectors, or by engineering restriction sites at specificlocations using PCR amplification [using the same conditions describedin (a) above]. In all cases, clones were verified by restrictionmapping, and, where indicated, DNA sequencing.

[0447] Protein preparations from induced cultures of each of theseconstructs were analyzed, by SDS-PAGE, to estimate protein stability(Coomassie Blue staining) and immunoreactivity against anti-toxin Bspecific antiserum (Western analysis). Higher levels of intact (i.e.,nondegraded), full length fusion proteins were observed with the smallerconstructs as compared with the larger recombinants, and a series ofexpression constructs spanning the entire toxin B gene were constructed(FIGS. 18, 19 and 20 and Table 23).

[0448] Constructs that expressed significant levels of recombinant toxinB protein (greater than 1 mg/liter intact recombinant protein) in E.coli were identified and a series of these clones that spans the toxin Bgene are shown in FIG. 19 and summarized in Table 23. These clones wereutilized to isolate pure toxin B recombinant protein from the entiretoxin B gene. Significant protein yields were obtained from pMALexpression constructs spanning the entire toxin B gene, and yields offull length soluble fusion protein ranged from an estimated 1 mg/literculture (pMB1100-1530) to greater than 20 mg/liter culture(pMB1750-2360).

[0449] Representative purifications of MBP and poly-histidine-taggedtoxin B recombinants are shown in FIGS. 21 and 22. FIG. 21 shows aCoomassie Blue stained 7.5% SDS-PAGE gel on which various proteinsamples extracted from bacteria harboring pMB1850-2360 wereelectrophoresed. Samples were loaded as follows: Lane 1: proteinextracted from uninduced culture; Lane 2: induced culture protein; Lane3: total protein from induced culture after sonication; Lane 4: solubleprotein; and Lane 5: eluted affinity purified protein. FIG. 22 depictsthe purification of recombinant proteins expressed in bacteria harboringeither pPB1850-2360 (Lanes 1-3) or pPB1750-2360 (Lanes 4-6). Sampleswere loaded as follows: uninduced total protein (Lanes 1 and 4); inducedtotal protein (Lanes 2 and 5); and eluted affinity purified protein(Lanes 3 and 6). The broad range molecular weight protein markers(BioRad) are shown in Lane 7.

[0450] Thus, although high level expression was not attained using largeexpression constructs from the toxin B gene, usable levels ofrecombinant protein were obtained by isolating induced protein from aseries of smaller pMAL expression constructs that span the entire toxinB gene.

[0451] These results represent the first demonstration of thefeasibility of expressing recombinant toxin B protein to high levels inE. coli. As well, expression of small regions of the putative ligandbinding domain (repeat region) of toxin B as native protein yieldedinsoluble protein, while large constructs, or fusions to MBP weresoluble (FIG. 19), demonstrating that specific methodologies arenecessary to produce soluble fusion protein from this interval.

[0452] iii) Clone Construction and Expression Details

[0453] A portion of the toxin B gene containing the toxin B repeatregion [amino acid residues 1852-2362 of toxin B (SEQ ID NO:20)] wasisolated by PCR amplification of this interval of the toxin B gene fromC. difficile genomic DNA. The sequence, and location within the toxin Bgene, of the two PCR primers [P7 (SEQ ID NO:13) and P8 (SEQ ID NO:14)]used to amplify this region are shown in FIG. 18.

[0454] DNA from the PCR amplification was purified by chloroformextraction and ethanol precipitation as described above. The DNA wasrestricted with SpeI, and the cleaved DNA was resolved by agarose gelelectrophoresis. The restriction digestion with SpeI cleaved the 3.6 kbamplification product into a 1.8 kb doublet band. This doublet band wascut from the gel and mixed with appropriately cut, gel purified pMALc orpET23b vector. These vectors were prepared by digestion with HindIII,filling in the overhanging ends using the Klenow enzyme, and cleavingwith XbaI (pMALc) or NheI (pET23b). The gel purified DNA fragments werepurified using the Prep-A-Gene kit (BioRad) and the DNA was ligated,transformed and putative recombinant clones analyzed by restrictionmapping.

[0455] pET and pMal clones containing the toxin B repeat insert (aainterval 1750-2360 of toxin B) were verified by restriction mapping,using enzymes that cleaved specific sites within the toxin B region. Inboth cases fusion of the toxin B SpeI site with either the compatibleXbaI site (pMal) or compatible NheI site (pET) is predicted to create anin frame fusion. This was confirmed in the case of the pMB1750-2360clone by DNA sequencing of the clone junction and 5′ end of the toxin Binsert using a MBP specific primer (New England Biolabs). In the case ofthe pET construct, the fusion of the blunt ended toxin B 3′ end to thefilled HindIII site should create an in-frame fusion with the C-terminalpoly-histidine sequence in this vector. The pPB1750-2360 clone selectedhad lost, as predicted, the HindIII site at this clone junction; thiseliminated the possibility that an additional adenosine residue wasadded to the 3′ end of the PCR product by a terminal transferaseactivity of the Pfu polymerase, since fusion of this adenosine residueto the filled HindIII site would regenerate the restriction site (andwas observed in several clones).

[0456] One liter cultures of each expression construct were grown, andfusion protein purified by affinity chromatography on either an amyloseresin column (pMAL constructs; resin supplied by New England Biolabs) orNi-chelate column (pET constructs; resin supplied by Qiagen or Novagen)as described [Williams et al. (1995), supra]. The integrity and purityof the fusion proteins were determined by Coomassie staining of SDS-PAGEprotein gels as well as Western blot analysis with either an affinitypurified goat polyclonal antiserum (Tech Lab), or a chicken polyclonalPEG prep, raised against the toxin B protein (CTB) as described above inExample 8. In both cases, affinity purification resulted in yields inexcess of 20 mg protein per liter culture, of which greater than 90% wasestimated to be full-length recombinant protein. It should be noted thatthe poly-histidine affinity tagged protein was released from the QiagenNi-NTA resin at low imidazole concentration (60 mM), necessitating theuse of a 40 mM imidazole rather than a 60 mM imidazole wash step duringpurification.

[0457] A periplasmically secreted version of pMB1750-2360 wasconstructed by replacement of the promoter and MBP coding region of thisconstruct with that from a related vector (pMAL™-p2; New EnglandBiolabs) in which a signal sequence is present at the N-terminus of theMBP, such that fusion protein is exported. This was accomplished bysubstituting a BglII-EcoRV promoter fragment from pMAL-p2 intopMB1750-2360. The yields of secreted, affinity purified protein(recovered from osmotic shock extracts as described by Riggs in CurrentProtocols in Molecular Biology, Vol. 2, Ausubel, et al., Eds. (1989),Current Protocols, pp. 16.6.1-16.6.14] from this vector (pMB1750-2360)were 6.5 mg/liter culture, of which 50% was estimated to be full-lengthfusion protein.

[0458] The interval was also expressed in two non-overlapping fragments.pMB1750-1970 was constructed by introduction of a frameshift intopMB1750-2360, by restriction with HindIII, filling in the overhangingends and religation of the plasmid. Recombinant clones were selected byloss of the HindIII site, and further restriction map analysis.Recombinant protein expression from this vector was more than 20mg/liter of greater than 90% pure protein.

[0459] The complementary region was expressed in pMB1970-2360. Thisconstruct was created by removal of the 1750-1970 interval ofpMB1750-2360. This was accomplished by restriction of this plasmid withEcoRI (in the pMalc polylinker 5′ to the insert) and III, filling in theoverhanging ends, and religation of the plasmid. The resultant plasmid,pMB1970-2360, was made using both intracellularly and secreted versionsof the pMB1750-2360 vector.

[0460] No fusion protein was secreted in the pMBp1970-2360 version,perhaps due to a conformational constraint that prevents export of thefusion protein. However, the intracellularly expressed vector producedgreater than 40 mg/liter of greater than 90% full-length fusion protein.

[0461] Constructs to precisely express the toxin B repeats in eitherpMalc (pMB1850-2360) or pET16b (pPB1850-2360) were constructed asfollows. The DNA interval including the toxin B repeats was PCRamplified as described above utilizing PCR primers P14 (SEQ ID NO:19)and P8 (SEQ ID NO:14). Primer P14 adds a EcoRI site immediately flankingthe start of the toxin B repeats.

[0462] The amplified fragment was cloned into the pT7 Blue T-vector(Novagen) and recombinant clones in which single copies of the PCRfragment were inserted in either orientation were selected(pT71850-2360) and confirmed by restriction mapping. The insert wasexcised from two appropriately oriented independently isolatedpT71850-2360 plasmids, with EcoRI (5′ end of repeats) and PstI (in theflanking polylinker of the vector), and cloned into EcoRI/PstI cleavedpMalc vector. The resulting construct (pMB1850-2360) was confirmed byrestriction analysis, and yielded 20 mg/l of soluble fusion protein[greater than 90% intact (i.e., nondegraded)] after affinitychromatography. Restriction of this plasmid with HindIII and religationof the vector resulted in the removal of the 1970-2360 interval. Theresultant construct (pMB1850-1970) expressed greater than 70 mg/liter of90% full length fusion protein.

[0463] The pPB1850-2360 construct was made by cloning a EcoRI (filledwith Klenow)-BamHI fragment from a pT71850-2360 vector (oppositeorientation to that used in the pMB1850-2360 construction) into NdeI(filled)/BamHI cleaved pET16b vector. Yields of affinity purifiedsoluble fusion protein were 15 mg/liter, of greater than 90% full lengthfusion protein.

[0464] Several smaller expression constructs from the 1750-2070 intervalwere also constructed in His-tagged pET vectors, but expression of theseplasmids in the BL21 (DE3) host resulted in the production of highlevels of mostly insoluble protein (see Table 23 and FIG. 19). Theseconstructs were made as follows.

[0465] pPB1850-1970 was constructed by cloning a BglII-HindIII fragmentof pPB1850-2360 into BglII/HindIII cleaved pET23b vector. pPB1850-2070was constructed by cloning a BglII-PvuII fragment of pPB1850-2360 intoBglII/HincII cleaved pET23b vector. pPB1750-1970(c) was constructed byremoval of the internal HindIII fragment of a pPB1750-2360 vector inwhich the vector HindIII site was regenerated during cloning (presumablyby the addition of an A residue to the amplified PCR product by terminaltransferase activity of Pfu polymerase). The pPB1750-1970(n) constructwas made by insertion of the insert containing the NdeI-HindIII fragmentof pPB1750-2360 into identically cleaved pETHisb vector. All constructswere confirmed by restriction digestion.

[0466] An expression construct that directs expression of the 10-470 aainterval of toxin B was constructed in the pMalc vector as follows. ANheI (a site 5′ to the insert in the pET23 vector)-AflII (filled)fragment of the toxin B gene from pPB10-1530 was cloned into XbaI(compatible with NheI)/HindIII (filled) pMalc vector. The integrity ofthe construct (pMB10-470) was verified by restriction mapping and DNAsequencing of the 5′ clone junction using a MBP specific DNA primer (NewEngland Biolabs). However, all expressed protein was degraded to the MBPmonomer MW.

[0467] A second construct spanning this interval (aa 10-470) wasconstructed by cloning the PCR amplification product from a reactioncontaining the P9 (SEQ ID NO:15) and P10 (SEQ ID NO:16) primers (FIG.18) into the pETHisa vector. This was accomplished by cloning the PCRproduct as an EcoRI-blunt fragment into EcoRI HincII restricted vectorDNA; recombinant clones were verified by restriction mapping. Althoughthis construct (pPB10⁻⁵²⁰) allowed expression and purification(utilizing the N-terminal polyhistidine affinity tag) of intact fusionprotein, yields were estimated at less than 500 μg per liter culture.

[0468] Higher yield of recombinant protein from this interval (aa10-520) were obtained by expression of the interval in two overlappingclones. The 10-330aa interval was cloned in both pETHisa and pMalcvectors, using the BamHI-AflII (filled) DNA fragment from pPB10-520.This fragment was cloned into BamHI-HindIII (filled) restricted pMalc orBamHI-HincII restricted pETHisa vector. Recombinant clones were verifiedby restriction mapping. High level expression of either insoluble (pET)or soluble (pMal) fusion protein was obtained. Total yields of fusionprotein from the pMB10-330 construct (FIG. 18) were 20 mg/liter culture,of which 10% was estimated to be full-length fusion protein. Althoughyields of this interval were higher and >90% full-length recombinantprotein produced when expressed from the pET construct, the pMal fusionwas utilized since the expressed protein was soluble and thus morelikely to have the native conformation.

[0469] The pMB260-520 clone was constructed by cloning EcoRI-XbaIcleaved amplified DNA from a PCR reaction containing the P11 (SEQ IDNO:17) and P10 (SEQ ID NO:16) DNA primers (FIG. 18) into similarlyrestricted pMalc vector.

[0470] Yields of affinity purified protein were 10 mg/liter, of whichapproximately 50% was estimated to be full-length recombinant protein.

[0471] The aa510-1110 interval was expressed as described below. Thisentire interval was expressed as a pMal fusion by cloning theNheI-HindIII fragment of pUCB10-1530 into XbaI-HindIII cleaved pMalcvector. The integrity of the construct (pMB510-1110) was verified byrestriction mapping and DNA sequencing of the 5′ clone junction using aMBP specific DNA primer. The yield of affinity purified protein was 25mg/liter culture, of which 5% was estimated to be full-length fusionprotein (1 mg/liter).

[0472] To attempt to obtain higher yields, this region was expressed intwo fragments (aa510-820, and 820-1110) in the pMalc vector. ThepMB510-820 clone was constructed by insertion of a SacI (in the pMalcpolylinker 5′ to the insert)-HpaI DNA fragment from pMB5110-110 intoSacI/StuI restricted pMalc vector. The pMB820-1110 vector wasconstructed by insertion of the HpaI-HindIII fragment of pUCB10-1530into BamHI (filled)/HindIII cleaved pMalc vector. The integrity of theseconstructs were verified by restriction mapping and DNA sequencing ofthe 5′ clone junction using a MBP specific DNA primer.

[0473] Recombinant protein expressed from the pMB510-820 vector washighly unstable. However, high levels (20 mg/liter) of >90% full-lengthfusion protein were obtained from the pMB820-1105 construct. Thecombination of partially degraded pMB510-1110 protein (enriched for the510-820 interval) with the pMB820-1110 protein provides usable amountsof recombinant antigen from this interval.

[0474] The aa1100-1750 interval was expressed as described below. Theentire interval was expressed in the pMalc vector from a construct inwhich the AccI(filled)-SpeI fragment of pPB10-1750 was inserted intoStuI/XbaI (XbaI is compatible with SpeI; StuI and filled AccI sites areboth blunt ended) restricted pMalc. The integrity of this construct(pMB1100-1750) was verified by restriction mapping and DNA sequencing ofthe clone junction using a MBP specific DNA primer. Although 15 mg/literof affinity purified protein was isolated from cells harboring thisconstruct, the protein was greater than 99% degraded to MBP monomersize.

[0475] A smaller derivative of pMB1100-1750 was constructed byrestriction of pMB1100-1750 with AflII and SalI (in the pMalc polylinker3′ to the insert), filling in the overhanging ends, and religating theplasmid. The resultant clone (verified by restriction digestion and DNAsequencing) has deleted the aa1530-1750 interval, was designatedpMB11100-1530. pMB1100-1530 expressed recombinant protein at a yield ofgreater than 40 mg/liter, of which 5% was estimated to be full-lengthfusion protein.

[0476] Three constructs were made to express the remaining interval.Initially, a BspHI (filled)-SpeI fragment from pPB10-1750 was clonedinto EcoRI(filled)/XbaI cleaved pMalc vector. The integrity of thisconstruct (pMB1570-1750) was verified by restriction mapping and DNAsequencing of the 5′ clone junction using a MBP specific DNA primer.Expression of recombinant protein from this plasmid was very low,approximately 3 mg affinity purified protein per liter, and most wasdegraded to MBP monomer size. This region was subsequently expressedfrom a PCR amplified DNA fragment. A PCR reaction utilizing primers P13[SEQ ID NO:18; P13 was engineered to introduce an EcoRI site 5′ toamplified toxin B sequences] and P8 (SEQ ID NO:14) was performed on C.difficile genomic DNA as described above. The amplified fragment wascleaved with EcoRI and SpeI, and cloned into EcoRI/XbaI cleaved pMalcvector. The resultant clone (pMB1530-1750) was verified by restrictionmap analysis, and recombinant protein was expressed and purified. Theyield was greater than 20 mg protein per liter culture and it wasestimated that 25% was full-length fusion protein; this was asignificantly higher yield than the original pMB1570-1750 clone: Theinsert of pMB1530-1750 (in a EcoRI-SalI fragment) was transferred to thepETHisa vector (EcoRI/XhoI cleaved, XhoI and SalI ends are compatible).No detectable fusion protein was purified on Ni-Chelate columns fromsoluble lysates of cells induced to express fusion protein from thisconstruct. TABLE 23 Summary Of Toxin B Expression Constructs^(a) Yield %Full Clone Affinity Tag (mg/liter) Length pPB10-1750 none low (estimatedfrom ? Western blot hyb.) pPB10-1530 none low (as above) ? pMB10-470 MBP15 mg/l    0%  pPB10-520 poly-his 0.5 mg/l   20% pPB10-330 poly-his >20mg/l   90% (insoluble) pMB10-330 MBP 20 mg/l   10% pMB260-520 MBP 10mg/l   50% pMB510-1110 MBP 25 mg/l     5%  pMB510-820 MBP degraded (byWestern blot hyb) pMB820-1110 MBP 20 mg/l   90% pMB1100-1750 MBP 15 mg/l   0%  pMB1100-1530 MBP 40 mg/l     5%  pMB1570-1750 MBP 3 mg/l  <5%pPB1530-1750 poly-his no purified ? protein detected pMB1530-1750 MBP 20mg/l   25% pMB1750-2360 MBP >20 mg/l   >90% pMBp1750-2360 MBP 6.5 mg/l  50% (secreted) pPB1750-2360 poly-his >20 mg/l >90% pMB1750-1970MBP >20 mg/l >90% pMB1970-2360 MBP 40 mg/l >90% pMBp1970-2360 MBP (nosecretion) NA pMB1850-2360 MBP 20 mg/l >90% pPB1850-2360 poly-his 15mg/l >90% pMB1850-1970 MBP 70 mg/l >90% pPB1850-1970 poly-his >10mg/l >90% (insoluble) pPB1850-2070 poly-his >10 22 mg/l >90% (insoluble)pPB1750-1970(c) poly-his >10 mg/l >90% (insoluble) pPB1750-1970(n)poly-his >10 mg/l >90% (insoluble)

Example 19 Identification, Purification and Induction of NeutralizingAntibodies Against Recombinant C. difficile Toxin B Protein

[0477] To determine whether recombinant toxin B polypeptide fragmentscan generate neutralizing antibodies, typically animals would firstbe-immunized with recombinant proteins and anti-recombinant antibodiesare generated. These anti-recombinant protein antibodies are then testedfor neutralizing ability in vivo or in vitro. Depending on theimmunogenic nature of the recombinant polypeptide, the generation ofhigh-titer antibodies against that protein may take several months. Toaccelerate this process and identify which recombinant polypeptide(s)may be the best candidate to generate neutralizing antibodies, depletionstudies were performed. Specifically, recombinant toxin B polypeptidewere pre-screened by testing whether they have the ability to bind toprotective antibodies from a CTB antibody preparation and hence depletethose antibodies of their neutralizing capacity. Those recombinantpolypeptides found to bind CTB, were then utilized to generateneutralizing antibodies. This Example involved: a) identification ofrecombinant sub-regions within toxin B to which neutralizing antibodiesbind; b) identification of toxin B sub-region specific antibodies thatneutralize toxin B in vivo; and c) generation and evaluation ofantibodies reactive to recombinant toxin B polypeptides.

[0478] a) Identification of Recombinant Sub-Regions within Toxin B toWhich Neutralizing Antibodies Bind

[0479] Sub-regions within toxin B to which neutralizing antibodies bindwere identified by utilizing recombinant toxin B proteins to depleteprotective antibodies from a polyclonal pool of antibodies againstnative C. difficile toxin B. An in vivo assay was developed to evaluateprotein preparations for the ability to bind neutralizing antibodies.Recombinant proteins were first pre-mixed with antibodies directedagainst native toxin B (CTB antibody; see Example 8) and allowed toreact for one hour at 37° C. Subsequently, C. difficile toxin B (CTB;Tech Lab) was added at a concentration lethal to hamsters and incubatedfor another hour at 37° C. After incubation this mixture was injectedintraperitoneally (IP) into hamsters. If the recombinant polypeptidecontains neutralizing epitopes, the CTB antibodies will lose its abilityto protect the hamsters against death from CTB. If partial or completeprotection occurs with the CTB antibody-recombinant mixture, thatrecombinant contains only weak or non-neutralizing epitopes of toxin B.This assay was performed as follows.

[0480] Antibodies against CTB were generated in egg laying Leghorn hensas described in Example 8. The lethal dosage (LD₁₀₀) of C. difficiletoxin B when delivered I.P. into 40 g female Golden Syrian hamsters(Charles River) was determined to be 2.5 to 5 μg. Antibodies generatedagainst CTB and purified by PEG precipitation could completely protectthe hamsters at the I.P. dosage determined above. The minimal amount ofCTB antibody needed to afford good protection against 5 μg of CTB wheninjected I.P. into hamsters was also determined (1×PEG prep). Theseexperiments defined the parameters needed to test whether a givenrecombinant protein could deplete protective CTB antibodies.

[0481] The cloned regions tested for neutralizing ability cover theentire toxin B gene and were designated as Intervals (INT) 1 through 5(see FIG. 19). Approximately equivalent final concentrations of eachrecombinant polypeptide were tested. The following recombinantpolypeptides were used: 1) a mixture of intervals 1 and 2 (INT-1, 2); 2)a mixture of Intervals 4 and 5 (INT-4, 5) and 3) Interval 3 (INT-3).Recombinant proteins (each at about 1 mg total protein) were firstpreincubated with a final CTB antibody concentration of 1× [i.e., pelletdissolved in original yolk volume as described in Example 1(c)] in afinal volume of 5 mls for 1 hour at 37° C. Twenty-five μg of CTB (at aconcentration of 5 μg/ml), enough CTB to kill 5 hamsters, was then addedand the mixture was then incubated for 1 hour at 37° C. Five, 40 gfemale hamsters (Charles River) in each treatment group were then eachgiven 1 ml of the mixture I.P. using a tuberculin syringe with a 27gauge needle. The results of this experiment are shown in Table 24.TABLE 24 Binding Of Neutralizing Antibodies By INT 3 Protein Number OfNumber Of Animals Animals Treatment Group¹ Alive Dead CTB antibodies 3 2CTB antibodies + INT1, 2 3 2 CTB antibodies + INT4, 5 3 2 CTBantibodies + INT 3 0 5

[0482] As shown in Table 24, the addition of recombinant proteins fromINT-1, 2 or INT-4, 5 had no effect on the in vivo protective ability ofthe CTB antibody preparation compared to the CTB antibody preparationalone. In contrast, INT-3 recombinant polypeptide was able to remove allof the toxin B neutralizing ability of the CTB antibodies asdemonstrated by the death of all the hamsters in that group.

[0483] The above experiment was repeated, using two smaller expressedfragments (pMB 1750-1970 and pMB1970-2360, see FIG. 19) comprising theINT-3 domain to determine if that domain could be further subdividedinto smaller neutralizing epitopes. In addition, full-length INT-3polypeptide expressed as a nickel tagged protein (pPB1750-2360) wastested for neutralizing ability and compared to the original INT-3expressed MBP fusion (pMB1750-2360). The results are shown in Table 25.TABLE 25 Removal Of Neutralizing Antibodies By Repeat ContainingProteins Number Of Number of Animals Animals Treatment Group¹ Alive DeadCTB antibodies 5 0 CTB antibodies + pPB1750-2360 0 5 CTB antibodies +pMB1750-2360 0 5 CTB antibodies + pMB1970-2360 3 2 CTB antibodies +pMB1750-1970 2 3

[0484] The results summarized in Table 25 indicate that the smallerpolypeptide fragments within the INT-3 domain, pMB1750-1970 andpMB1970-2360, partially lose the ability to bind to and removeneutralizing antibodies from the CTB antibody pool. These resultsdemonstrate that the full length INT-3 polypeptide is required tocompletely deplete the CTB antibody pool of neutralizing antibodies.This experiment also shows that the neutralization epitope of INT-3 canbe expressed in alternative vector systems and the results areindependent of the vector utilized or the accompanying fusion partner.

[0485] Other Interval 3 specific proteins were subsequently tested forthe ability to remove neutralizing antibodies within the CTB antibodypool as described above. The Interval 3 specific proteins used in thesestudies are summarized in FIG. 23. In FIG. 23 the followingabbreviations are used: pP refers to the pET23 vector; pM refers to thepMALc vector; B refers to toxin B; the numbers refer to the amino acidinterval expressed in the clone. The solid black ovals represent theMBP; and HHH represents the poly-histidine tag.

[0486] Only recombinant proteins comprising the entire toxin B repeatdomain (pMB1750-2360, pPB1750-2360 and pPB1850-2360) can bind andcompletely remove neutralizing antibodies from the CTB antibody pool.Recombinant proteins comprising only a portion of the toxin B repeatdomain were not capable of completely removing neutralizing antibodiesfrom the CTB antibody pool (pMB1750-1970 and pMB1970-2360 couldpartially remove neutralizing antibodies while pMB1850-1970 andpPB1850-2070 failed to remove any neutralizing antibodies from the CTBantibody pool).

[0487] The above results demonstrate that only the complete ligandbinding domain (repeat region) of the toxin B gene can bind andcompletely remove neutralizing antibodies from the CTB antibody pool.These results demonstrate that antibodies directed against the entiretoxin B repeat region are necessary for in vivo toxin neutralization(see FIG. 23; only the recombinant proteins expressed by thepMB1750-2360, pPB1750-2360 and pPB1850-2360 vectors are capable ofcompletely removing the neutralizing antibodies from the CTB antibodypool).

[0488] These results represent the first indication that the entirerepeat region of toxin B would be necessary for the generation ofantibodies capable of neutralizing toxin B, and that sub-regions may notbe sufficient to generate maximal titers of neutralizing antibodies.

[0489] b) Identification of Toxin B Sub-Region Specific Antibodies ThatNeutralize Toxin B In Vivo

[0490] To determine if antibodies directed against the toxin B repeatregion are sufficient for neutralization, region specific antibodieswithin the CTB antibody preparation were affinity purified, and testedfor in vivo neutralization. Affinity columns containing recombinanttoxin B repeat proteins were made as described below. A separateaffinity column was prepared using each of the following recombinanttoxin B repeat proteins: pPB1750-2360, pPB1850-2360, pMB1750-1970 andpMB1970-2360.

[0491] For each affinity column to be made, four ml of PBS-washedActigel resin (Sterogene) was coupled overnight at room temperature with5-10 mg of affinity purified recombinant protein (first extensivelydialyzed into PBS) in 15 ml tubes (Falcon) containing a {fraction(1/10)} final volume Ald-coupling solution (1 M sodiumcyanoborohydride). Aliquots of the supernatants from the couplingreactions, before and after coupling, were assessed by Coomassiestaining of 7.5% SDS-PAGE gels. Based on protein band intensities, inall cases greater than 30% coupling efficiencies were estimated. Theresins were poured into 10 ml columns (BioRad), washed extensively withPBS, pre-eluted with 4M guanidine-HCl (in 10 mM Tris-HCl, pH 8.0) andreequilibrated in PBS. The columns were stored at 4° C.

[0492] Aliquots of a CTB IgY polyclonal antibody preparation (PEG prep)were affinity purified on each of the four columns as described below.The columns were hooked to a UV monitor (ISCO), washed with PBS and 40ml aliquots of a 2×PEG prep (filter sterilized using a 0.45μ filter)were applied. The columns were washed with PBS until the baseline valuewas re-established. The columns were then washed with BBStween to elutenonspecifically binding antibodies, and reequilibrated with PBS. Boundantibody was eluted from the column in 4M guanidine-HCl (in 10 mMTris-HCl, pH8.0). The eluted antibody was immediately dialyzed against a100-fold excess of PBS at 4° C. for 2 hrs. The samples were thendialyzed extensively against at least 2 changes of PBS, and affinitypurified antibody was collected and stored at 4° C. The antibodypreparations were quantified by UV absorbance. The elution volumes werein the range of 4-8 ml. All affinity purified stocks contained similartotal antibody concentrations, ranging from 0.25-0.35% of the totalprotein applied to the columns.

[0493] The ability of the affinity purified antibody preparations toneutralize toxin B in vivo was determined using the assay outlined in a)above. Affinity purified antibody was diluted 1:1 in PBS before testing.The results are shown in Table 26.

[0494] In all cases similar levels of toxin neutralization was observed,such that lethality was delayed in all groups relative to preimmunecontrols. This result demonstrates that antibodies reactive to therepeat region of the toxin B gene are sufficient to neutralize toxin Bin vivo. The hamsters will eventually die in all groups, but this deathis maximally delayed with the CTB PEG prep antibodies. Thusneutralization with the affinity purified (AP) antibodies is not ascomplete as that observed with the CTB prep before affinitychromatography. This result may be due to loss of activity duringguanidine denaturation (during the elution of the antibodies from theaffinity column) or the presence of antibodies specific to other regionsof the toxin B gene that can contribute to toxin neutralization (presentin the CTB PEG prep). TABLE 26 Neutralization Of Toxin B By AffinityPurified Antibodies Number Number Animals Animals Treatment group^(a)Alive^(b) Dead^(b) Preimmune¹ 0 5 CTB¹; 400 μg 5 0 CTB (AP onpPB1750-2360);² 875 μg 5 0 CTB (AP on pMB1750-1970);² 875 μg 5 0 CTB (APon pMB1970-2360);² 500 μg 5 0

[0495] The observation that antibodies affinity purified against thenon-overlapping pMB1750-1970 and pMB1970-2360 proteins neutralized toxinB raised the possibility that either 1) antibodies specific to repeatsub-regions are sufficient to neutralize toxin B or 2) sub-regionspecific proteins can bind most or all repeat specific antibodiespresent in the CTB polyclonal pool. This would likely be due toconformational similarities between repeats, since homology in theprimary amino acid sequences between different repeats is in the rangeof only 25-75% [Eichel-Streiber, et al. (1992) Molec. Gen. Genetics233:260]. These possibilities were tested by affinity chromatography.

[0496] The CTB PEG prep was sequentially depleted 2× on the pMB1750-1970column; only a small elution peak was observed after the secondchromatography, indicating that most reactive antibodies were removed.This interval depleted CTB preparation was then chromatographed on thepPB1850-2360 column; no antibody bound to the column. The reactivity ofthe CTB and CTB (pMB1750-1970 depleted) preps to pPB1750-2360,pPB1850-2360, pMB1750-1970 and pMB1970-2360 proteins was then determinedby ELISA using the protocol described in Example 13(c). Briefly, 96-wellmicrotiter plates (Falcon, Pro-Bind Assay Plates) were coated withrecombinant protein by adding 100 μl volumes of protein at 1-2 μg/ml inPBS containing 0.005% thimerosal to each well and incubating overnightat 4° C. The next morning, the coating suspensions were decanted and thewells were washed three times using PBS. In order to block non-specificbinding sites, 100 μl of 1.0% BSA (Sigma) in PBS (blocking solution) wasthen added to each well, and the plates were incubated for 1 hr. at 37°C. The blocking solution was decanted and duplicate samples of 150 μl ofdiluted antibody was added to the first well of a dilution series. Theinitial testing serum dilution was ({fraction (1/200)} for CTB prep,(the concentration of depleted CTB was standardized by OD₂₈₀) inblocking solution containing 0.5% Tween 20, followed by 5-fold serialdilutions into this solution. This was accomplished by seriallytransferring 30 μl aliquots to 120 μl buffer, mixing, and repeating thedilution into a fresh well. After the final dilution, 30 μl was removedfrom the well such that all wells contained 120 μl final volume. A totalof 5 such dilutions were performed (4 wells total). The plates wereincubated for 1 hr at 37° C. Following this incubation, the seriallydiluted samples were decanted and the wells were washed three timesusing PBS containing 0.5% Tween 20 (PBST), followed by two 5 min washesusing BBS-Tween and a final three washes using PBST. To each well, 100μl of {fraction (1/1000)} diluted secondary antibody [rabbitanti-chicken IgG alkaline phosphatase (Sigma) diluted in blockingsolution containing 0.5% Tween 20] was added, and the plate wasincubated 1 hr at 37° C. The conjugate solutions were decanted and theplates were washed 6 times in PBST, then once in 50 mM Na₂CO₃, 10 mMMgCl₂, pH 9.5. The plates were developed by the addition of 100 μl of asolution containing 1 mg/ml para-nitro phenyl phosphate (Sigma)dissolved in 50 mM Na₂CO₃, 10 mM MgCl₂, pH9.5 to each well. The plateswere then incubated at room temperature in the dark for 5-45 min. Theabsorbency of each well was measured at 410 nm using a Dynatech MR 700plate reader.

[0497] As predicted by the affinity chromatography results, depletion ofthe CTB prep on the pMB1750-1970 column removed all detectablereactivity to the pMB1970-2360 protein. The reciprocal purification of aCTB prep that was depleted on the pMB1970-2360 column yielded no boundantibody when chromatographed on the pMB1750-1970 column. These resultsdemonstrate that all repeat reactive antibodies in the CTB polyclonalpool recognize a conserved structure that is present in non-overlappingrepeats. Although it is possible that this conserved structurerepresents rare conserved linear epitopes, it appears more likely thatthe neutralizing antibodies recognize a specific protein conformation.This conclusion was also suggested by the results of Western blothybridization analysis of CTB reactivity to these recombinant proteins.

[0498] Western blots of 7.5% SDS-PAGE gels, loaded and electrophoresedwith defined quantities of each recombinant protein, were probed withthe CTB polyclonal antibody preparation. The blots were prepared anddeveloped with alkaline phosphatase as described in Example 3. Theresults are shown in FIG. 24.

[0499]FIG. 24 depicts a comparison of immunoreactivity of IgY antibodyraised against either native or recombinant toxin B antigen. Equalamounts of pMB1750-1970 (lane 1), pMB1970-2360 (lane 2), pPB1850-2360(lane 3) as well as a serial dilution of pPB1750-2360 (lanes 4-6comprising 1×, {fraction (1/10)}× and {fraction (1/100)}× amounts,respectively) proteins were loaded in duplicate and resolved on a 7.5%SDS-PAGE gel. The gel was blotted and each half was hybridized with PEGprep IgY antibodies from chickens immunized with either native CTB orpPB1750-2360 protein. Note that the full-length pMB1750-1970 protein wasidentified only by antibodies reactive to the recombinant protein(arrows).

[0500] Although the CTB prep reacts with the pPB1750-2360, pPB1850-2360,and pMB1970-2360 proteins, no reactivity to the pMB1750-1970 protein wasobserved (FIG. 24). Given that all repeat reactive antibodies can bebound by this protein during affinity chromatography, this resultindicates that the protein cannot fold properly on Western blots. Sincethis eliminates all antibody reactivity, it is unlikely that the repeatreactive antibodies in the CTB prep recognize linear epitopes. This mayindicate that in order to induce protective antibodies, recombinanttoxin B protein will need to be properly folded.

[0501] c) Generation and Evaluation of Antibodies Reactive toRecombinant Toxin B Polypeptides

[0502] i) Generation of Antibodies Reactive to Recombinant Toxin BProteins

[0503] Antibodies against recombinant proteins were generated in egglaying Leghorn hens as described in Example 13. Antibodies were raised[using Freunds adjuvant (Gibco) unless otherwise indicated] against thefollowing recombinant proteins: 1) a mixture of Interval 1+2 proteins(see FIG. 18); 2) a mixture of interval 4 and 5 proteins (see FIG. 18);3) pMB1970-2360 protein; 4) pPB1750-2360 protein; 5) pMB1750-2360; 6)pMB1750-2360 [Titermax adjuvant (Vaxcell)]; 7) pMB1750-2360 [Gerbuadjuvant (Biotech)]; 8) pMBp1750-2360 protein; 9) pPB1850-2360; and 10)pMB1850-2360.

[0504] Chickens were boosted at least 3 times with recombinant proteinuntil ELISA reactivity [using the protocol described in b) above withthe exception that the plates were coated with pPB1750-2360 protein] ofpolyclonal PEG preps was at least equal to that of the CTB polyclonalantibody PEG prep. ELISA titers were determined for the PEG preps fromall of the above immunogens and were found to be comparable ranging from1:12500 to 1:62500. High titers were achieved in all cases except in 6)pMB1750-2360 in which strong titers were not observed using the Titermaxadjuvant, and this preparation was not tested further.

[0505] ii) Evaluation of Antibodies Reactive to Recombinant Proteins byWestern Blot Hybridization

[0506] Western blots of 7.5% SDS-PAGE gels, loaded and electrophoresedwith defined quantities of recombinant protein (pMB1750-1970,pPB1850-2360, and pMB1970-2360 proteins and a serial dilution of thepPB1750-2360 to allow quantification of reactivity), were probed withthe CTB, pPB1750-2360, pMB1750-2360 and pMB1970-2360 polyclonal antibodypreparations (from chickens immunized using Freunds adjuvant). The blotswere prepared and developed with alkaline phosphatase as described abovein b).

[0507] As shown in FIG. 24, the CTB and pMB1970-2360 preps reactedstrongly with the pPB1750-2360, pPB1850-2360, and pMB1970-2360 proteinswhile the pPB1750-2360 and pMB1970-2360 (Gerbu) preparations reactedstrongly with all four proteins. The Western blot reactivity of thepPB1750-2360 and pMB1970-2360 (Gerbu) preparations were equivalent tothat of the CTB preparation, while reactivity of the pMB1970-2360preparation was <10% that of the CTB prep. Despite equivalent ELISAreactivities only weak reactivity (approximately 1%) to the recombinantproteins were observed in PEG preps from two independent groupsimmunized with the pMB1750-2360 protein and one group immunized with thepMB1750-2360 preparation using Freunds adjuvant.

[0508] Affinity purification was utilized to determine if thisdifference in immunoreactivity by Western blot analysis reflectsdiffering antibody titers. Fifty ml 2× PEG preparations from chickensimmunized with either pMB1750-2360 or pMB1970-2360 protein werechromatographed on the pPB1750-2360 affinity column from b) above, asdescribed. The yield of affinity purified antibody (% total protein inpreparation) was equivalent to the yield obtained from a CTB PEGpreparation in b) above. Thus, differences in Western reactivity reflecta qualitative difference in the antibody pools, rather than quantitativedifferences., These results demonstrate that certain recombinantproteins are more effective at generating high affinity antibodies (asassayed by Western blot hybridization).

[0509] iii) In Vivo Neutralization of Toxin B Using Antibodies Reactiveto Recombinant Protein

[0510] The in vivo hamster model [described in Examples 9 and 14(b)] wasutilized to assess the neutralizing ability of antibodies raised againstrecombinant toxin B proteins. The results from three experiments areshown below in Tables 27-29.

[0511] The ability of each immunogen to neutralize toxin B in vivo hasbeen compiled and is shown in Table 30. As predicted from therecombinant protein-CTB premix studies (Table 24) only antibodies toInterval 3 (1750-2366) and not the other regions of toxin B (i.e.,intervals 1-5) are protective. Unexpectedly, antibodies generated toINT-3 region expressed in pMAL vector (pMB1750-2360 and pMB1750-2360)using Freunds adjuvant were non-neutralizing. This observation isreproducible, since no neutralization was observed in two independentimmunizations with pMB1750-2360 and one immunization with pMB1750-2360.The fact that 5× quantities of affinity purified toxin B repeat specificantibodies from pMB1750-2360 PEG preps cannot neutralize toxin B while1× quantities of affinity purified anti-CTB antibodies can (Table 28)demonstrates that the differential ability of CTB antibodies toneutralize toxin B is due to qualitative rather than quantitativedifferences in these antibody preparations. Only when this region wasexpressed in an alternative vector (pPB1750-2360) or using analternative adjuvant with the pMB1750-2360 protein were neutralizingantibodies generated. Importantly, antibodies raised using Freundsadjuvant to pPB1850-2360, which contains a fragment that is only 100amino acids smaller than recombinant pPB1750-2360, are unable toneutralize toxin B in vivo (Table 27); note also that the same vector isused for both pPB1850-2360 and pPB1750-2360. TABLE 27 In VivoNeutralization Of Toxin B Number Number Animals Animals TreatmentGroup^(a) Alive^(b) Dead^(b) Preimmune 0 5 CTB 5 0 INT 1 + 2 0 5 INT 4 +5 0 5 pMB1750-2360 0 5 pMB1970-2360 0 5 pPB1750-2360 5 0

[0512] TABLE 28 In Vivo Neutralization Of Toxin B Using AffinityPurified Antibodies Number Number Animals Animals Treatment Group^(a)Alive^(b) Dead^(b) Preimmune(1) 0 5 CTB(1) 5 0 pPB1750-2360(1) 5 0 1.5mg anti-pMB1750-2360(2) 1 4 1.5 mg anti-pMB1970-2360(2) 0 5 300 uganti-CTB(2) 5 0

[0513] TABLE 29 Generation Of Neutralizing Antibodies Utilizing TheGerbu Adjuvant Number Number Animals Animals Treatment Group^(a)Alive^(b) Dead^(b) Preimmune 0 5 CTB 5 0 pMB1970-2360 0 5 pMB1850-2360 05 pPB1850-2360 0 5 pMB1750-2360 5 0 (Gerbu adj)

[0514] TABLE 30 In Vivo Neutralization Of Toxin B Tested In vivo Prepar-Antigen Neutraliz- Immunogen Adjuvant ation^(a) Utilized For APation^(b) Preimmune NA¹ PEG NA no CTB (native) Titermax PEG NA yes CTB(native) Titermax AP pPB1750-2360 yes CTB (native) Titermax APpPB1850-2360 yes CTB (native) Titermax AP pPB1750-1970 yes CTB (native)Titermax AP pPB1970-2360 yes pMB1750-2360 Freunds PEG NA no pMB1750-2360Freunds AP pPB1750-2360 no pMB1750-2360 Gerbu PEG NA yes pMB1970-2360Freunds PEG NA no pMB1970-2360 Freunds AP pPB1750-2360 no pPB1750-2360Freunds PEG NA yes pPB1850-2360 Freunds PEG NA no pMB1850-2360 FreundsPEG NA no INT 1 + 2 Freunds PEG NA no INT 4 + 5 Freunds PEG NA no

[0515] The pPB1750-2360 antibody pool confers significant in vivoprotection, equivalent to that obtained with the affinity purified CTBantibodies. This correlates with the observed high affinity of thisantibody pool (relative to the pMB1750-2360 or pMB1970-2360 pools) asassayed by Western blot analysis (FIG. 24). These results provide thefirst demonstration that in vivo neutralizing antibodies can be inducedusing recombinant toxin B protein as immunogen.

[0516] The failure of high concentrations of antibodies raised againstthe pMB1750-2360 protein (using Freunds adjuvant) to neutralize, whilethe use of Gerbu adjuvant and pMB1750-2360 protein generates aneutralizing response, demonstrates that conformation or presentation ofthis protein is essential for the induction of neutralizing antibodies.These results are consistent with the observation that the neutralizingantibodies produced when native CTB is used as an immunogen appear torecognize conformational epitopes [see section b) above]. This is thefirst demonstration that the conformation or presentation of recombinanttoxin B protein is essential to generate high titers of neutralizingantibodies.

Example 20 Determination of Quantitative and Qualitative DifferencesBetween pMB1750-2360, pMB1750-2360 (Gerbu) or pPB1750-2360 IgYPolyclonal Antibody Preparations

[0517] In Example 19, it was demonstrated that toxin B neutralizingantibodies could be generated using specific recombinant toxin Bproteins (pPB1750-2360) or specific adjuvants. Antibodies raised againstpMB1750-2360 were capable of neutralizing the enterotoxin effect oftoxin B when the recombinant protein was used to immunize hens inconjunction with the Gerbu adjuvant, but not when Freunds adjuvant wasused. To determine the basis for these antigen and adjuvantrestrictions, toxin B-specific antibodies present in the neutralizingand non-neutralizing PEG preparations were isolated by affinitychromatography and tested for qualitative or quantitative differences.The example involved a) purification of anti-toxin B specific antibodiesfrom pMB1750-2360 and pPB1750-2360 PEG preparations and b) in vivoneutralization of toxin B using the affinity purified antibody.

[0518] a) Purification of Specific Antibodies From pMB1750-2360 andpPB1750-2360 PEG Preparations

[0519] To purify and determine the concentration of specific antibodies(expressed as the percent of total antibody) within the pPB1750-2360(Freunds and Gerbu) and pPB1750-2360 PEG preparations, definedquantities of these antibody preparations were chromatographed on anaffinity column containing the entire toxin B repeat region(pPB1750-2360). The amount of affinity purified antibody was thenquantified.

[0520] An affinity column containing the recombinant toxin B repeatprotein, pPB1750-2360, was made as follows. Four ml of PBS-washedActigel resin (Sterogene) was coupled with 5 mg of pPB1750-2360 affinitypurified protein (dialyzed into PBS; estimated to be greater than 95%full length fusion protein) in a 15 ml tube (Falcon) containing{fraction (1/10)} final volume Ald-coupling solution (1M sodiumcyanoborohydride). Aliquots of the supernatant from the couplingreactions, before and after coupling, were assessed by Coomassiestaining of 7.5% SDS-PAGE gels. Based on protein band intensities,greater than 95% (approximately 5 mg) of recombinant protein was coupledto the resin. The coupled resin was poured into a 10 ml column (BioRad),washed extensively with PBS, pre-eluted with 4M guanidine-HCl (in 10 mMTris-HCl, pH 8.0; 0.005% thimerosal) and re-equilibrated in PBS andstored at 4° C.

[0521] Aliquots of pMB1750-2360, pMB1750-2360 (Gerbu) or pPB1750-2360IgY polyclonal antibody preparations (PEG preps) were affinity purifiedon the above column as follows. The column was attached to an UV monitor(ISCO), and washed with PBS. Forty ml aliquots of 2×PEG preps (filtersterilized using a 0.45μ filter and quantified by OD₂₈₀ beforechromatography) was applied. The column was washed with PBS until thebaseline was re-established (the column flow-through was saved), washedwith BBSTween to elute nonspecifically binding antibodies andre-equilibrated with PBS. Bound antibody was eluted from the column in4M guanidine-HCl (in 10 mM Tris-HCL, pH 8.0, 0.005% thimerosal) and theentire elution peak collected in a 15 ml tube (Falcon). The column wasre-equilibrated, and the column eluate re-chromatographed as describedabove. The antibody preparations were quantified by UV absorbance (theelution buffer was used to zero the spectrophotometer). Approximately 10fold higher concentrations of total purified antibody was obtained uponelution of the first chromatography pass relative to the second pass.The low yield from the second chromatography pass indicated that most ofthe specific antibodies were removed by the first round ofchromatography.

[0522] Pools of affinity purified specific antibodies were prepared bydialysis of the column elutes after the first column chromatography passfor the pMB1750-2360, pMB1750-2360 (Gerbu) or pPB1750-2360 IgYpolyclonal antibody preparations. The elutes were collected on ice andimmediately dialyzed against a 100-fold volume of PBS at 4° C. for 2hrs. The samples were then dialyzed against 3 changes of a 65-foldvolume of PBS at 4° C. Dialysis was performed for a minimum of 8 hrs perchange of PBS. The dialyzed samples were collected, centrifuged toremove insoluble debris, quantified by OD₂₈₀, and stored at 4° C.

[0523] The percentage of toxin B repeat-specific antibodies present ineach preparation was determined using the quantifications of antibodyyields from the first column pass (amount of specific antibody recoveredafter first pass/total protein loaded). The yield of repeat-specificaffinity purified antibody (expressed as the percent of total protein inthe preparation) in: 1) the pMB1750-2360 PEG prep was approximately0.5%, 2) the pMB1750-2360 (Gerbu) prep was approximately 2.3%, and 3)the pPB1750-2360 prep was approximately 0.4%. Purification of a CTB IgYpolyclonal antibody preparation on the same column demonstrated that theconcentration of toxin B repeat specific antibodies in the CTBpreparation was 0.35%.

[0524] These results demonstrate that 1) the use of Gerbu adjuvantenhanced the titer of specific antibody produced against thepMB1750-2360 protein 5-fold relative to immunization using Freundsadjuvant, and 2) the differences seen in the in vivo neutralizationability of the pMB1750-2360 (not neutralizing) and pPB1750-2360(neutralizing) and CTB (neutralizing) PEG preps seen in Example 19 wasnot due to differences in the titers of repeat-specific antibodies inthe three preparations because the titer of repeat-specific antibody wassimilar for all three preps; therefore the differing ability of thethree antibody preparations to neutralize toxin B must reflectqualitative differences in the induced toxin B repeat-specificantibodies. To confirm that qualitative differences exist betweenantibodies raised in hens immunized with different recombinant proteinsand/or different adjuvants, the same amount of affinity purifiedanti-toxin B repeat (aa 1870-2360 of toxin B) antibodies from thedifferent preparations was administered to hamsters using the in vivohamster model as described below.

[0525] b) In Vivo Neutralization of Toxin B Using Affinity PurifiedAntibody

[0526] The in vivo hamster model was utilized to assess the neutralizingability of the affinity purified antibodies raised against recombinanttoxin B proteins purified in (a) above. As well, a 4×IgY PEG preparationfrom a second independent immunization utilizing the pPB1750-2360antigen with Freunds adjuvant was tested for in vivo neutralization. Theresults are shown in Table 31.

[0527] The results shown in Table 31 demonstrate that:

[0528] 1) as shown in Example 19 and reproduced here, 1.5 mg of affinitypurified antibody from pMB1750-2360 immunized hens using Freundsadjuvant does not neutralize toxin B in vivo. However, 300 μg ofaffinity purified antibody from similarly immunized hens utilizing Gerbuadjuvant demonstrated complete neutralization of toxin B in vivo. Thisdemonstrates that Gerbu adjuvant, in addition to enhancing the titer ofantibodies reactive to the pMB1750-2360 antigen relative to Freundsadjuvant (demonstrated in (a) above), also enhances the yield ofneutralizing antibodies to this antigen, greater than 5 fold.

[0529] 2) Complete in vivo neutralization of toxin B was observed with1.5 mg of affinity purified antibody from hens immunized withpPB1750-2360 antigen, but not with pMB1750-2360 antigen, when Freundsadjuvant was used. This demonstrates, using standardized toxin Brepeat-specific antibody concentrations, that neutralizing antibodieswere induced when pPB1750-2360 but not pMB1750-2360 was used as theantigen with Freunds adjuvant.

[0530] 3) Complete in vivo neutralization was observed with 300 μg ofpMB1750-2360 (Gerbu) antibody, but not with 300 μg of pPB1750-2360(Freunds) antibody. Thus the pMB1750-2360 (Gerbu) antibody has a highertiter of neutralizing antibodies than the pPB1750-2360 (Freunds)antibody.

[0531] 4) Complete neutralization of toxin B was observed using 300 μgof CTB antibody [affinity purified (AP)] but not 100 μg CTB antibody (APor PEG prep). This demonstrates that greater than 100 μg of toxin Brepeat-specific antibody (anti-CTB) is necessary to neutralize 25 μgtoxin B in vivo in this assay, and that affinity purified antibodiesspecific to the toxin B repeat interval neutralize toxin B aseffectively as the PEP prep of IgY raised against the entire CTB protein(shown in this assay).

[0532] 5) As was observed with the initial pPB1750-2360 (IgY) PEGpreparation (Example 19), complete neutralization was observed with aIgY PEG preparation isolated from a second independent group ofpPB1750-2360 (Freunds) immunized hens. This demonstrates thatneutralizing antibodies are reproducibly produced when hens areimmunized with pPB1750-2360 protein utilizing Freunds adjuvant. TABLE 31In vivo Neutralization Of Toxin B Using Affinity Purified AntibodiesNumber Number Animals Animals Treatment Group^(a) Alive^(b) Dead^(b)Preimmune¹ 0 5 CTB (300 μg)² 5 0 CTB (100 μg)² 1 4 pMB1750-2360 (G) (5mg)² 5 0 pMB1750-2360 (G) (1.5 mg)² 5 0 pMB1750-2360 (G) (300 μg)² 5 0pMB1750-2360 (F) (1.5 mg)² 0 5 pPB1750-2360 (F) (1.5 mg)² 5 0pPB1750-2360 (F) (300 μg)² 1 4 CTB (100 μg)³ 2 3 pPB1750-2360 (F) (500μg)¹ 5 0

Example 21 Diagnostic Enzyme Immunoassays for C. difficile Toxins A andB

[0533] The ability of the recombinant toxin proteins and antibodiesraised against these recombinant proteins (described in the aboveexamples) to form the basis of diagnostic assays for the detection ofclostridial toxin in a sample was examined. Two immunoassay formats weretested to quantitatively detect C. difficile toxin A and toxin B from abiological specimen. The first format involved a competitive assay inwhich a fixed amount of recombinant toxin A or B was immobilized on asolid support (e.g., microtiter plate wells) followed by the addition ofa toxin-containing biological specimen mixed with affinity-purified orPEG fractionated antibodies against recombinant toxin A or B. If toxinis present in a specimen, this toxin will compete with the immobilizedrecombinant toxin protein for binding to the anti-recombinant antibodythereby reducing the signal obtained following the addition of areporter reagent. The reporter reagent detects the presence of antibodybound to the immobilized toxin protein.

[0534] In the second format, a sandwich immunoassay was developed usingaffinity-purified antibodies to recombinant toxin A and B. Theaffinity-purified antibodies to recombinant toxin A and B were used tocoat microtiter wells instead of the recombinant polypeptides (as wasdone in the competitive assay format). Biological samples containingtoxin A or B were then added to the wells followed by the addition of areporter reagent to detect the presence of bound toxin in the well.

[0535] a) Competitive Immunoassay for the Detection of C. difficileToxin

[0536] Recombinant toxin A or B was attached to a solid support bycoating 96 well microtiter plates with the toxin protein at aconcentration of 1 μg/ml in PBS. The plates were incubated overnight at2-8° C. The following morning, the coating solutions were removed andthe remaining protein binding sites on the wells were blocked by fillingeach well with a PBS solution containing 0.5% BSA and 0.05% Tween-20.Native C. difficile toxin A or B (Tech Lab) was diluted to 4 μg/ml instool extracts from healthy Syrian hamsters (Sasco). The stool extractswere made by placing fecal pellets in a 15 ml centrifuge tube; PBS wasadded at 2 ml/pellet and the tube was vortexed to create a uniformsuspension. The tube was then centrifuged at 2000 rpm for 5 min at roomtemperature. The supernatant was removed; this comprises the stoolextract. Fifty ill of the hamster stool extract was pipetted into eachwell of the microtiter plates to serve as the diluent for serialdilutions of the 4 μg/ml toxin samples. One hundred μl of the toxinsamples at 4 μg/ml was pipetted into the first row of wells in themicrotiter plate, and 50 μl aliquots were removed and diluted seriallydown the plate in duplicate. An equal volume of affinity purifiedanti-recombinant toxin antibodies [1 ng/well of anti-pMA1870-2680antibody was used for the detection of toxin A; 0.5 ng/well ofanti-pMB1750-2360(Gerbu) was used for the detection of toxin B] wereadded to appropriate wells, and the plates were incubated at roomtemperature for 2 hours with gentle agitation. Wells serving as negativecontrol contained antibody but no native toxin to compete for binding.

[0537] Unbound toxin and antibody were removed by washing the plates 3to 5 times with PBS containing 0.05% Tween-20. Following the wash step,100 μl of rabbit anti-chicken IgG antibody conjugated to alkalinephosphatase (Sigma) was added to each well and the plates were incubatedfor 2 hours at room temperature. The plates were then washed as beforeto remove unbound secondary antibody. Freshly prepared alkalinephosphatase substrate (1 mg/ml p-nitrophenyl phosphate (Sigma) in 50 mMNa₂CO₃, pH 9.5; 10 mM MgCl₂) was added to each well. Once sufficientcolor developed, the plates were read on a Dynatech MR700 microtiterplate reader using a 410 nm filter.

[0538] The results are summarized in Tables 32 and 33. For the resultsshown in Table 32, the wells were coated with recombinant toxin Aprotein (pMA 1870-2680).

[0539] The amount of native toxin A added (present as an addition tosolubilized hamster stool) to a given well is indicated (0 to 200 ng).Antibody raised against the recombinant toxin A protein, pMA1870-2680,was affinity purified on the an affinity column containing pPA1870-2680(described in Example 20). As shown in Table 32, the recombinant toxin Aprotein and affinity-purified antitoxin can be used for the basis of acompetitive immunoassay for the detection of toxin A in biologicalsamples.

[0540] Similar results were obtained using the recombinant toxin B,pPB1750-2360, and antibodies raised against pMB1750-2360(Gerbu). For theresults shown in Table 33, the wells were coated with recombinant toxinB protein (pPB1750-2360). The amount of native toxin B added (present asan addition to solubilized hamster stool) to a given well is indicated(0 to 200 ng). Antibody raised against the recombinant toxin B protein,pMB1750-2360(Gerbu), was affinity purified on the an affinity columncontaining pPB1850-2360 (described in Example 20). As shown in Table 33,the recombinant toxin B protein and affinity-purified antitoxin can beused for the basis of a competitive immunoassay for the detection oftoxin B in biological samples.

[0541] In this competition assay, the reduction is consideredsignificant over the background levels at all points; therefore theassay can be used to detect samples containing less than 12.5 ng toxinA/well and as little as 50-100 ng toxin B/well. TABLE 32 CompetitiveInhibition Of Anti-C. difficile Toxin A By Native Toxin A ng ToxinA/Well OD₄₁₀ Readout 200 0.176 100 0.253 50 0.240 25 0.259 12.5 0.3096.25 0.367 3.125 0.417 0 0.590

[0542] TABLE 33 Competitive Inhibition Of Anti-C. difficile Toxin B ByNative Toxin B ng Toxin B/Well OD₄₁₀ Readout 200 0.392 100 0.566 500.607 25 0.778 12.5 0.970 6.25 0.902 3.125 1.040 0 1.055

[0543] These competitive inhibition assays demonstrate that native C.difficile toxins and recombinant C. difficile toxin proteins can competefor binding to antibodies raised against recombinant C. difficile toxinsdemonstrating that these anti-recombinant toxin antibodies provideeffective diagnostic reagents.

[0544] b) Sandwich Immunoassay for the Detection of C. difficile Toxin

[0545] Affinity-purified antibodies against recombinant toxin A or toxinB were immobilized to 96 well microtiter plates as follows. The wellswere passively coated overnight at 4° C. with affinity purifiedantibodies raised against either pMA1870-2680 (toxin A) orpMB1750-2360(Gerbu) (toxin B). The antibodies were affinity purified asdescribed in Example 20. The antibodies were used at a concentration of1 μg/ml and 100 μl was added to each microtiter well. The wells werethen blocked with 200 μl of 0.5% BSA for 2 hours at room temperature andthe blocking solution was then decanted. Stool samples from healthySyrian hamsters were resuspended in PBS, pH 7.4 (2 ml PBS/stool pelletwas used to resuspend the pellets and the sample was centrifuged asdescribed above). The stool suspension was then spiked with native C.difficile toxin A or B (Tech Lab) at 4 μg/ml. The stool suspensionscontaining toxin (either toxin A or toxin B) were then serially dilutedtwo-fold in stool suspension without toxin and 50 μl was added induplicate to the coated microtiter wells. Wells containing stoolsuspension without toxin served as the negative control.

[0546] The plates were incubated for 2 hours at room temperature andthen were washed three times with PBS. One hundred μl of either goatanti-native toxin A or goat anti-native toxin B (Tech Lab) diluted1:1000 in PBS containing 1% BSA and 0.05% Tween 20 was added to eachwell. The plates were incubated for another 2 hours at room temperature.The plates were then washed as before and 100 μl of alkalinephosphatase-conjugated rabbit anti-goat IgG (Cappel, Durham, N.C.) wasadded at a dilution of 1:1000. The plates were incubated for another 2hours at room temperature. The plates were washed as before thendeveloped by the addition of 100 μl/well of a substrate solutioncontaining 1 mg/ml p-nitrophenyl phosphate (Sigma) in 50 mM Na₂CO₃, pH9.5; 10 mM MgCl₂. The absorbance of each well was measured using a platereader (Dynatech) at 410 nm. The assay results are shown in Tables 34and 35. TABLE 34 C. difficile Toxin A Detection In Stool UsingAffinity-Purified Antibodies Against Toxin A ng Toxin A/Well OD₄₁₀Readout 200 0.9 100 0.8 50 0.73 25 0.71 12.5 0.59 6.25 0.421 0 0

[0547] TABLE 35 C. difficile Toxin B Detection In Stool UsingAffinity-Purified Antibodies Against Toxin B ng Toxin B/Well OD₄₁₀Readout 200 1.2 100 0.973 50 0.887 25 0.846 12.5 0.651 6.25 0.431 00.004

[0548] The results shown in Tables 34 and 35 show that antibodies raisedagainst recombinant toxin A and toxin B fragments can be used to detectthe presence of C. difficile toxin in stool samples. These antibodiesform the basis for a sensitive sandwich immunoassay which is capable ofdetecting as little as 6.25 ng of either toxin A or B in a 50 μl stoolsample. As shown above in Tables 34 and 35, the background for thissandwich immunoassay is extremely low; therefore, the sensitivity ofthis assay is much lower than 6.25 ng toxin/well. It is likely thattoxin levels of 0.5 to 1.0 pg/well could be detected by this assay.

[0549] The results shown above in Tables 32-35 demonstrate clear utilityof the recombinant reagents in C. difficile toxin detection systems.

Example 22 Construction and Expression of C. botulinum C Fragment FusionProteins

[0550] The C. botulinum type A neurotoxin gene has been cloned andsequenced [Thompson, et al., Eur. J. Biochem. 189:73 (1990)]. Thenucleotide sequence of the toxin gene is available from the EMBL/GenBanksequence data banks under the accession number X52066; the nucleotidesequence of the coding region is listed in SEQ ID NO:27. The amino acidsequence of the C. botulinum type A neurotoxin is listed in SEQ IDNO:28. The type A neurotoxin gene is synthesized as a single polypeptidechain which is processed to form a dimer composed of a light and a heavychain linked via disulfide bonds. The 50 kD carboxy-terminal portion ofthe heavy chain is referred to as the C fragment or the H_(C) domain.

[0551] Previous attempts by others to express polypeptides comprisingthe C fragment of C. botulinum type A toxin as a native polypeptide(e.g., not as a fusion protein) in E. coli have been unsuccessful [H. F.LaPenotiere, et al. in Botulinum and Tetanus Neurotoxins, DasGupta, Ed.,Plenum Press, New York (1993), pp. 463-466]. Expression of the Cfragment as a fusion with the E. coli MBP was reported to result in theproduction of insoluble protein (H. F. LaPenotiere, et al., supra).

[0552] In order to produce soluble recombinant C fragment proteins in Ecoli, fusion proteins comprising a synthetic C fragment gene derivedfrom the C. botulinum type A toxin and either a portion of the C.difficile toxin protein or the MBP were constructed. This exampleinvolved a) the construction of plasmids encoding C fragment fusionproteins and b) expression of C. botulinum C fragment fusion proteins inE. coli.

[0553] a) Construction of Plasmids Encoding C Fragment Fusion Proteins

[0554] In Example 11, it was demonstrated that the C. difficile toxin Arepeat domain can be efficiently expressed and purified in E. coli aseither native (expressed in the pET 23a vector in clone pPA1870-2680) orfusion (expressed in the pMALc vector as a fusion with the E. coli MBPin clone pMA1870-2680) proteins. Fusion proteins comprising a fusionbetween the MBP, portions of the C. difficile toxin A repeat domain(shown to be expressed as a soluble fusion protein) and the C fragmentof the C. botulinum type A toxin were constructed. A fusion proteincomprising the C fragment of the C. botulinum type A toxin and the MBPwas also constructed.

[0555]FIG. 25 provides a schematic representation of the botulinalfusion proteins along with the donor constructs containing the C.difficile toxin A sequences or C botulinum C fragment sequences whichwere used to generate the botulinal fusion proteins. In FIG. 25, thesolid boxes represent C. difficile toxin A gene sequences, the openboxes represent C. botulinum C fragment sequences and the solid blackovals represent the E. coli MBP. When the name for a restriction enzymeappears inside parenthesis, this indicates that the restriction site wasdestroyed during construction. An asterisk appearing with the name for arestriction enzyme indicates that this restriction site was recreated atthe cloning junction.

[0556] In FIG. 25, a restriction map of the pMA1870-2680 andpPA1100-2680 constructs (described in Example 11) which containsequences derived from the C. difficile toxin A repeat domain are shown;these constructs were used as the source of C. difficile toxin A genesequences for the construction of plasmids encoding fusions between theC. botulinum C fragment gene and the C. difficile toxin A gene. ThepMA1870-2680 expression construct expresses high levels of soluble,intact fusion protein (20 mg/liter culture) which can be affinitypurified on an amylose column (purification described in Example 11d).

[0557] The pAlterBot construct (FIG. 25) was used as the source of C.botulinum C fragment gene sequences for the botulinal fusion proteins.pAlterBot was obtained from J. Middlebrook and R. Lemley at the U.S.Department of Defense. pAlterBot contains a synthetic C. botulinum Cfragment inserted in to the pALTER-1® vector (Promega). This synthetic Cfragment gene encodes the same amino acids as does the naturallyoccurring C fragment gene. The naturally occurring C fragment sequences,like most clostridial genes, are extremely A/T rich (Thompson et al.,supra). This high A/F content creates expression difficulties in E. coliand yeast due to altered codon usage frequency and fortuitouspolyadenylation sites, respectively. In order to improve the expressionof C fragment proteins in E. coli, a synthetic version of the gene wascreated in which the non-preferred codons were replaced with preferredcodons.

[0558] The nucleotide sequence of the C. botulinum C fragment genesequences contained within pAlterBot is listed in SEQ ID NO:22. Thefirst six nucleotides (ATGGCT) encode a methionine and alanine residue,respectively. These two amino acids result from the insertion of the C.botulinum C fragment sequences into the pALTER® vector and provide theinitiator methionine residue. The amino acid sequence of the C.botulinum C fragment encoded by the sequences contained within pAlterBotis listed in SEQ ID NO:23. The first two amino acids (Met Ala) areencoded by vector-derived sequences. From the third amino acid residueonward (Arg), the amino acid sequence is identical to that found in theC. botulinum type A toxin gene.

[0559] The pMA1870-2680, pPA1100-2680 and pAlterBot constructs were usedas progenitor plasmids to make expression constructs in which fragmentsof the C. difficile toxin A repeat domain were expressed as geneticfusions with the C. botulinum C fragment gene using the pMAL-cexpression vector (New England BioLabs). The pMAL-c expression vectorgenerates fusion proteins which contain the MBP at the amino-terminalend of the protein. A construct, pMBot, in which the C. botulinum Cfragment gene was expressed as a fusion with only the MBP wasconstructed (FIG. 25). Fusion protein expression was induced from E.coli strains harboring the above plasmids, and induced protein wasaffinity purified on an amylose resin column.

[0560] i) Construction of pBlueBot

[0561] In order to facilitate the cloning of the C. botulinum C fragmentgene sequences into a number of desired constructs, the botulinal genesequences were removed from pAlterBot and were inserted into thepBluescript plasmid (Stratagene) to generate pBlueBot (FIG. 25).pBlueBot was constructed as follows. Bacteria containing the pAlterBotplasmid were grown in medium containing tetracycline and plasmid DNA wasisolated using the QIAprep-spin Plasmid Kit (Qiagen). One microgram ofpAlterBot DNA was digested with NcoI and the resulting 3′ recessedsticky end was made blunt using the Klenow fragment of DNA polymerase I(here after the Klenow fragment). The pAlterBot DNA was then digestedwith HindIII to release the botulinal gene sequences (the Bot insert) asa blunt (filled NcoI site)-HindIII fragment. pBluescript vector DNA wasprepared by digesting 200 ng of pBluescript DNA with SmaI and HindIII.The digestion products from both plasmids were resolved on an agarosegel. The appropriate fragments were removed from the gel, mixed andpurified utilizing the Prep-a-Gene kit (BioRad). The eluted DNA was thenligated using T4 DNA ligase and used to transform competent DH5α cells(Gibco-BRL). Host cells were made competent for transformation using thecalcium chloride protocol of Sambrook et al., supra at 1.82-1.83.Recombinant clones were isolated and confirmed by restriction digestionusing standard recombinant molecular biology techniques (Sambrook et al,supra). The resultant clone, pBlueBot, contains several useful uniquerestriction sites flanking the Bot insert (i.e., the C. botulinum Cfragment sequences derived from pAlterBot) as shown in FIG. 25.

[0562] ii) Construction of C. difficile/C. botulinum/MBP Fusion Proteins

[0563] Constructs encoding fusions between the C. difficile toxin A geneand the C. botulinum C fragment gene and the MBP were made utilizing thesame recombinant DNA methodology outlined above; these fusion proteinscontained varying amounts of the C. difficile toxin A repeat domain.

[0564] The pMABot clone contains a 2.4 kb insert derived from the C.difficile toxin A gene fused to the Bot insert (i.e, the C. botulinum Cfragment sequences derived from pAlterBot). pMABot (FIG. 25) wasconstructed by mixing gel-purified DNA from NotI/HindIII digestedpBlueBot (the 1.2 kb Bot fragment), SpeI/NotI digested pPA1100-2680 (the2.4 kb C. difficile toxin A repeat fragment) and XbaI/HindIII digestedpMAL-c vector. Recombinant clones were isolated, confirmed byrestriction digestion and purified using the QIAprep-spin Plasmid Kit(Qiagen). This clone expresses the toxin A repeats and the botulinal Cfragment protein sequences as an in-frame fusion with the MBP.

[0565] The pMCABot construct contains a 1.0 kb insert derived from theC. difficile toxin A gene fused to the Bot insert (i.e, the C. botulinumC fragment sequences derived from pAlterBot). pMCABot was constructed bydigesting the pMABot clone with EcoRI to remove the 5′ end of the C.difficile toxin A repeat (see FIG. 25, the pMAL-c vector contains aEcoRI site 5′ to the C. difficile insert in the pMABot clone). Therestriction sites were filled and religated together after gelpurification. The resultant clone (pMCABot, FIG. 25) generated anin-frame fusion between the MBP and the remaining 3′ portion of the C.difficile toxin A repeat domain fused to the Bot gene.

[0566] The pMNABot clone contains the 1 kb SpeI/EcoRI (filled) fragmentfrom the C. difficile toxin A repeat domain (derived from clonepPA1100-2680) and the 1.2 kb C. botulinum C fragment gene as a NcoI(filled)/HindIII fragment (derived from pAlterBot). These two fragmentswere inserted into the pMAL-c vector digested with XbaI/HindIII. The twoinsert fragments were generated by digestion of the appropriate plasmidwith EcoRI (pPA1100-2680) or NcoI (pAlterBot) followed by treatment withthe Klenow fragment. After treatment with the Klenow fragment, theplasmids were digested with the second enzyme (either SpeI or HindIII).All three fragments were gel purified, mixed and Prep-a-Gene purifiedprior to ligation. Following ligation and transformation, putativerecombinants were analyzed by restriction analysis; the EcoRI site wasfound to be regenerated at the fusion junction, as was predicted for afusion between the filled EcoRI and NcoI sites.

[0567] A construct encoding a fusion protein between the botulinal Cfragment gene and the MBP gene was constructed (i.e., this fusion lacksany C. difficile toxin A gene sequences) and termed pMBot. The pMBotconstruct was made by removal of the C. difficile toxin A sequences fromthe pMABot construct and fusing the C fragment gene sequences to theMBP. This was accomplished by digestion of pMABot DNA with StuI (locatedin the pMALc polylinker 5′ to the XbaI site) and XbaI (located 3′ to theNotI site at the toxA-Bot fusion junction), filling in the XbaI siteusing the Klenow fragment, gel purifying the desired restrictionfragment, and ligating the blunt ends to circularize the plasmid.Following ligation and transformation, putative recombinants wereanalyzed by restriction mapping of the Bot insert (i.e, the C. botulinumC fragment sequences).

[0568] b) Expression of C. botulinum C Fragment Fusion Proteins in E.coli

[0569] Large scale (1 liter) cultures of the pMAL-c vector, and eachrecombinant construct described above in (a) were grown, induced, andsoluble protein fractions were isolated as described in Example 18. Thesoluble protein extracts were chromatographed on amylose affinitycolumns to isolate recombinant fusion protein. The purified recombinantfusion proteins were analyzed by running samples on SDS-PAGE gelsfollowed by Coomassie staining and by Western blot analysis as described[Williams et al, (1994) supra]. In brief, extracts were prepared andchromatographed in column buffer (10 mM NAPO₄, 0.5 M NaCl, 10 mMβ-mercaptoethanol, pH 7.2) over an amylose resin (New England Biolabs)column, and eluted with column buffer containing 10 mM maltose asdescribed [Williams, et al. (1994), supra). An SDS-PAGE gel containingthe purified protein samples stained with Coomassie blue is shown inFIG. 26.

[0570] In FIG. 26, the following samples were loaded. Lanes 1-6 containprotein purified from E. coli containing the pMAL-c, pPA1870-2680,pMABot, pMNABot, pMCABot and pMBot plasmids, respectively. Lane 7contains broad range molecular weight protein markers (BioRad).

[0571] The protein samples were prepared for electrophoresis by mixing 5μl of eluted protein with 5 μl of 2×SDS-PAGE sample buffer (0.125 mMTris-HCl, pH 6.8, 2 mM EDTA, 6% SDS, 20% glycerol, 0.025% bromophenolblue; β-mercaptoethanol is added to 5% before use). The samples wereheated to 95° C. for 5 min, then cooled and loaded on a 7.5% agaroseSDS-PAGE gel. Broad range molecular weight protein markers were alsoloaded to allow estimation of the MW of identified fusion proteins.After electrophoresis, protein was detected generally by staining thegel with Coomassie blue.

[0572] In all cases the yields were in excess of 20 mg fusion proteinper liter culture (see Table 36) and, with the exception of the pMCABotprotein, a high percentage (i.e., greater than 20-50% of total elutedprotein) of the eluted fusion protein was of a MW predicted for the fulllength fusion protein (FIG. 26). It was estimated (by visual inspection)that less than 10% of the pMCABot fusion protein was expressed as thefull length fusion protein. TABLE 36 Yield Of Affinity Purified C.botulinum C Fragment/MBP Fusion Proteins Yield Percentage Of TotalConstruct (mg/liter of Culture) Soluble Protein pMABot 24 5.0 pMCABot 345.0 pMNABot 40 5.5 pMBot 22 5.0 pMA1870-2680 40 4.8

[0573] These results demonstrate that high level expression of intact C.botulinum C fragment/C. difficile toxin A fusion proteins in E. coli isfeasible using the pMAL-c expression system. These results are incontrast to those reported by H. F. LaPenotiere, et al. (1993), supra.In addition, these results show that it is not necessary to fuse thebotulinal C fragment gene to the C. difficile toxin A gene in order toproduce a soluble fusion protein using the pMAL-c system in E. coli.

[0574] In order to determine whether the above-described botulinalfusion proteins were recognized by anti-C. botulinum toxin A antibodies,Western blots were performed. Samples containing affinity-purifiedproteins from E. coli containing the pMABot, pMCABot, pMNABot, pMBot,pMA1870-2680 or pMALc plasmids were analyzed. SDS-PAGE gels (7.5%acrylamide) were loaded with protein samples purified from eachexpression construct. After electrophoresis, the gels were blotted andprotein transfer was confirmed by Ponceau S staining (as described inExample 12b).

[0575] Following protein transfer, the blots were blocked by incubationfor 1 hr at 20° C. in blocking buffer [PBST (PBS containing 0.1% Tween20 and 5% dry milk)]. The blots were then incubated in 10 ml of asolution containing the primary antibody; this solution comprised a{fraction (1/500)} dilution of an anti-C. botulinum toxin A IgY PEG prep(described in Example 3) in blocking buffer. The blots were incubatedfor 1 hr at room temperature in the presence of the primary antibody.The blots were washed and developed using a rabbit anti-chicken alkalinephosphatase conjugate (Boehringer Mannheim) as the secondary antibody asfollows. The rabbit anti-chicken antibody was diluted to 1 μg/ml inblocking buffer (10 ml final volume per blot) and the blots wereincubated at room temperature for 1 hour in the presence of thesecondary antibody. The blots were then washed successively with PBST,BBS-Tween and 50 mM Na₂CO₃, pH 9.5. The blots were then developed infreshly-prepared alkaline phosphatase substrate buffer (100 μg/ml nitroblue tetrazolium, 50 μg/ml 5-bromo-chloro-indolylphosphate, 5 mM MgCl₂in 50 mM Na₂CO₃, pH 9.5). Development was stopped by flooding the blotswith distilled water and the blots were air dried.

[0576] This Western blot analysis detected anti-C. botulinum toxinreactive proteins in the pMABot, pMCABot, pMNABot and pMBot proteinsamples (corresponding to the predicted full length proteins identifiedabove by Coomassie staining in FIG. 26), but not in the pMA1100-2680 orpMALc protein samples.

[0577] These results demonstrate that the relevant fusion proteinspurified on an amylose resin as described above in section a) containedimmunoreactive C. botulinum C fragment protein as predicted.

Example 23 Generation of Neutralizing Antibodies by Nasal Administrationof pMBot Protein

[0578] The ability of the recombinant botulinal toxin proteins producedin Example 22 to stimulate a systemic immune response against botulinaltoxin epitopes was assessed. This example involved: a) the evaluation ofthe induction of serum IgG titers produced by nasal or oraladministration of botulinal toxin-containing C. difficile toxin A fusionproteins and b) the in vivo neutralization of C. botulinum type Aneurotoxin by anti-recombinant C. botulinum C fragment antibodies.

[0579] a) Evaluation of the Induction of Serum IgG Titers Produced byNasal or Oral Administration of Botulinal Toxin-Containing C. difficileToxin A Fusion Proteins

[0580] Six groups containing five 6 week old CF female rats (CharlesRiver) per group were immunized nasally or orally with one of thefollowing three combinations using protein prepared in Example 22: (1)250 μg pMBot protein per rat (nasal and oral); 2) 250 μg pMABot proteinper rat (nasal and oral); 3) 125 μg pMBot admixed with 125 μgpMA1870-2680 per rat (nasal and oral). A second set of 5 groupscontaining 3 CF female rats/group were immunized nasally or orally withone of the following combinations (4) 250 μg pMNABot protein per rat(nasal and oral) or 5) 250 μg pMAL-c protein per rat (nasal and oral).

[0581] The fusion proteins were prepared for immunization as follows.The proteins (in column buffer containing 10 mM maltose) were diluted in0.1 M carbonate buffer, pH 9.5 and administered orally or nasally in a200 μl volume. The rats were lightly sedated with ether prior toadministration. The oral dosing was accomplished using a 20 gaugefeeding needle. The nasal dosing was performed using a P-200micro-pipettor (Gilson). The rats were boosted 14 days after the primaryimmunization using the techniques described above and were bled 7 dayslater. Rats from each group were lightly etherized and bled from thetail. The blood was allowed to clot at 37° C. for 1 hr and the serum wascollected.

[0582] The serum from individual rats was analyzed using an ELISA todetermine the anti-C. botulinum type A toxin IgG serum titer. The ELISAprotocol used is a modification of that described in Example 13c.Briefly, 96-well microtiter plates (Falcon, Pro-Bind Assay Plates) werecoated with C. botulinum type A toxoid (prepared as described in Example3a) by placing 100 μl volumes of C. botulinum type A toxoid at 2.5 μg/mlin PBS containing 0.005% thimerosal in each well and incubatingovernight at 4° C. The next morning, the coating suspensions weredecanted and all wells were washed three times using PBS.

[0583] In order to block non-specific binding sites, 100 μl of blockingsolution [0.5% BSA in PBS] was then added to each well and the plateswere incubated for 1 hr at 37° C. The blocking solution was decanted andduplicate samples of 150 μl of diluted rat serum added to the first wellof a dilution series. The initial testing serum dilution was 1:30 inblocking solution containing 0.5% Tween 20 followed by 5-fold dilutionsinto this solution. This was accomplished by serially transferring 30 μlaliquots to 120 μl blocking solution containing 0.5% Tween 20, mixing,and repeating the dilution into a fresh well. After the final dilution,30 μl was removed from the well such that all wells contained 120 μlfinal volume. A total of 3 such dilutions were performed (4 wellstotal). The plates were incubated 1 hr at 37° C. Following thisincubation, the serially diluted samples were decanted and the wellswere washed six times using PBS containing 0.5% Tween 20 (PBST). To eachwell, 100 μl of a rabbit anti-Rat IgG alkaline phosphatase (Sigma)diluted ({fraction (1/1000)}) in blocking buffer containing 0.5% Tween20 was added and the plate was incubated for 1 hr at 37° C. Theconjugate solutions were decanted and the plates were washed asdescribed above, substituting 50 mM Na₂CO₃, pH 9.5 for the PBST in thefinal wash. The plates were developed by the addition of −100 μl of asolution containing 1 mg/ml para-nitro phenyl phosphate (Sigma)dissolved in 50 mM Na₂CO₃, 10 mM MgCl₂, pH 9.5 to each well, andincubating the plates at room temperature in the dark for 5-45 min. Theabsorbency of each well was measured at 410 nm using a Dynatech MR 700plate reader. The results are summarized in Tables 37 and 38 andrepresent mean serum reactivities of individual mice. TABLE 37Determination Of Anti - C. botulinum Type A Toxin Serum IgG TitersFollowing Immunization With C. botulinum C Fragment-Containing FusionProteins Nasal Oral Route of Immunization pMBot & pMBot& ImmunogenPREIMMUNE pMBot pMA1870-2680 pMABot pMBot pMA1870-2680 pMABot Dilution1:30 0.080 1.040 1.030 0.060 0.190 0.080 0.120 1:150 0.017 0.580 0.5400.022 0.070 0.020 0.027 1:750 0.009 0.280 0.260 0.010 0.020 0.010 0.0141:3750 0.007 0.084 0.090 0.009 0.009 0.010 0.007 # Rats 5 5 5 5 2 2Tested

[0584] TABLE 38 Determination Of Anti - C. botulinum Type A Toxin SerumIgG Titers Following Immunization With C. botulinum CFragment-Containing Fusion Proteins Route of Immunization PRE- NasalOral Immunogen IMMUNE pMBot pMABot pMNABot pMNABot Dilution 1:30 0.0400.557 0.010 0.015 0.010 1:150 0.009 0.383 0.001 0.003 0.002 1:750 0.0010.140 0.000 0.000 0.000 1:3750 0.000 0.040 0.000 0.000 0.000 # RatsTested 1 1 3 3

[0585] The above ELISA results demonstrate that reactivity against thebotulinal fusion proteins was strongest when the route of administrationwas nasal; only weak responses were stimulated when the botulinal fusionproteins were given orally. Nasally delivered pMbot and pMBot admixedwith pMA1870-2680 invoked the greatest serum IgG response. These resultsshow that only the pMBot protein is necessary to induce this response,since the addition of the pMA1870-2680 protein did not enhance antibodyresponse (Table 37). Placement of the C. difficile toxin A fragmentbetween the MBP and the C. botulinum C fragment protein dramaticallyreduced anti-bot IgG titer (see results using pMABot, pMCABot andpMNABot proteins).

[0586] This study demonstrates that the pMBot protein induces a strongserum IgG response directed against C. botulinum type A toxin whennasally administered.

[0587] b) In Vivo Neutralization of C. botulinum Type A Neurotoxin byAnti-Recombinant C. botulinum C Fragment Antibodies

[0588] The ability of the anti-C. botulinum type A toxin antibodiesgenerated by nasal administration of recombinant botulinal fusionproteins in rats (Example 22) to neutralize C. botulinum type A toxinwas tested in a mouse neutralization model. The mouse model is the artaccepted method for detection of botulinal toxins in body fluids and forthe evaluation of anti-botulinal antibodies [E. J. Schantz and D. A.Kautter, J. Assoc. Off. Anal. Chem. 61:96 (1990) and Investigational NewDrug (BB-IND-3703) application by the Surgeon General of the Departmentof the Army to the Federal Food and Drug Administration]. The anti-C.botulinum type A toxin antibodies were prepared as follows.

[0589] Rats from the group given pMBot protein by nasal administrationwere boosted a second time with 250 μg pMBot protein per rat and serumwas collected 7 days later. Serum from one rat from this group and froma preimmune rat was tested for anti-C. botulinum type A toxinneutralizing activity in the mouse neutralization model described below.

[0590] The LD₅₀ of a solution of purified C. botulinum type A toxincomplex, obtained from Dr. Eric Johnson (University of WisconsinMadison), was determined using the intraperitoneal (IP) method ofSchantz and Kautter [J. Assoc. Off. Anal. Chem. 61:96 (1978)] using18-22 gram female ICR mice and was found to be 3500 LD₅₀ ml. Thedetermination of the LD₅₀ was performed as follows. A Type A toxinstandard was prepared by dissolving purified type A toxin complex in 25mM sodium phosphate buffer, pH 6.8 to yield a stock toxin solution of3.15×10⁷LD₅₀/mg. The OD₂₇₈ of the solution was determined and theconcentration was adjusted to 10-20 μg/ml. The toxin solution was thendiluted 1:100 in gel-phosphate (30 mM phosphate, pH 6.4; 0.2% gelatin).Further dilutions of the toxin solution were made as shown below inTable 39. Two mice were injected IP with 0.5 ml of each dilution shownand the mice were observed for symptoms of botulism for a period of 72hours. TABLE 39 Determination Of The LD₅₀ Of Purified C. botulinum TypeA Toxin Complex Dilution Number Dead At 72 hr 1:320 2/2 1:640 2/2 1:12802/2 1:2560 0/2 (sick after 72 hr) 1:5120 0/2 (no symptoms)

[0591] From the results shown in Table 39, the toxin titer was assumedto be between 2560 LD₅₀/ml and 5120 LD₅₀/ml (or about 3840 LD₅₀/ml).This value was rounded to 3500 LD₅₀/ml for the sake of calculation.

[0592] The amount of neutralizing antibodies present in the serum ofrats immunized nasally with pMBot protein was then determined. Serumfrom two rats boosted with pMBot protein as described above andpreimmune serum from one rat was tested as follows. The toxin standardwas diluted 1:100 in gel-phosphate to a final concentration of 350LD₅₀/ml. One milliliter of the diluted toxin standard was mixed with 25μl of serum from each of the three rats and 0.2 ml of gel-phosphate. Themixtures were incubated at room temperature for 30 min with occasionalmixing. Each of two mice were injected with IP with 0.5 ml of themixtures. The mice were observed for signs of botulism for 72 hr. Micereceiving serum from rats immunized with pMBot protein neutralized thischallenge dose. Mice receiving preimmune rat serum died in less than 24hr.

[0593] The amount of neutralizing anti-toxin antibodies present in theserum of rats immunized with pMBot protein was then quantitated. Serumantibody titrations were performed by mixing 0.1 ml of each of theantibody dilutions (see Table 40) with 0.1 ml of a 1:10 dilution ofstock toxin solution (3.5×10⁴ LD₅₀/ml) with 1.0 ml of gel-phosphate andinjecting 0.5 ml IP into 2 mice per dilution. The mice were thenobserved for signs of botulism for 3 days (72 hr). The results aretabulated in Table 39.

[0594] As shown in Table 40 pMBot serum neutralized C. botulinum type Atoxin complex when used at a dilution of 1:320 or less. A meanneutralizing value of 168 IU/ml was obtained for the pMBot serum (an IUis defined as 10,000 mouse LD₅₀). This value translates to a circulatingserum titer of about 3.7 IU/mg of serum protein. This neutralizing titeris comparable to the commercially available bottled concentrated(Connaught Laboratories, Ltd.) horse anti-C. botulinum antiserum. A 10ml vial of Connaught antiserum contains about 200 mg/ml of protein; eachml can neutralize 750 IU of C. botulinum type A toxin. Afteradministration of one vial to a human, the circulating serum titer ofthe Connaught preparation would be approximately 25 IU/ml assuming anaverage serum volume of 3 liters). Thus, the circulating anti-C.botulinum titer seen in rats nasally immunized with pMBot protein (168IU/ml) is 6.7 time higher than the necessary circulation titer ofanti-C. botulinum antibody needed to be protective in humans. TABLE 40Quantitation Of Neutralizing Antibodies In pMBot Sera pMBot^(a) DilutionRat 1 Rat 2 1:20 2/2 2/2 1:40 2/2 2/2 1:80 2/2 2/2  1:160 2/2 2/2  1:320 2/2^(b)  2/2^(b)  1:640 0/2 0/2  1:1280 0/2 0/2  1:2560 0/2 0/2

[0595] These results demonstrate that antibodies capable of neutralizingC. botulinum type A toxin are induced when recombinant C. botulinum Cfragment fusion protein produced in E. coli is used as an immunogen.

Example 24 Production of Soluble C. botulinum C Fragment ProteinSubstantially Free of Endotoxin Contamination

[0596] Example 23 demonstrated that neutralizing antibodies aregenerated by immunization with the pMBot protein expressed in E. coli.These results showed that the pMBot fusion protein is a good vaccinecandidate. However, immunogens suitable for use as vaccines should bepyrogen-free in addition to having the capability of inducingneutralizing antibodies. Expression clones and conditions thatfacilitate the production of C. botulinum C fragment protein forutililization as a vaccine were developed.

[0597] The example involved: (a) determination of pyrogen content of thepMBot protein; (b) generation of C. botulinum C fragment protein free ofthe MBP; (c) expression of C. botulinum C fragment protein using variousexpression vectors; and (d) purification of soluble C. botulinum Cfragment protein substantially free of significant endotoxincontamination.

[0598] a) Determination of the Pyrogen Content of the pMBot Protein

[0599] In order to use a recombinant antigen as a vaccine in humans orother animals, the antigen preparation must be shown to be free ofpyrogens. The most significant pyrogen present in preparations ofrecombinant proteins produced in gram-negative bacteria, such as E.coli, is endotoxin [F. C. Pearson, Pyrogens: endotoxins, LAL testing anddepyrogentaion, (1985) Marcel Dekker, New York, pp. 23-56]. To evaluatethe utility of the pMBot protein as a vaccine candidate, the endotoxincontent in MBP fusion proteins was determined.

[0600] The endotoxin content of recombinant protein samples was assayedutilizing the Limulus assay (LAL kit; Associates of Cape Cod) accordingto the manufacturer's instructions. Samples of affinity-purified pMal-cprotein and pMA1870-2680 were found to contain high levels of endotoxin[>50,000 EU/mg protein; EU (endotoxin unit)]. This suggested that MBP-or toxin A repeat-containing fusions with the botulinal C fragmentshould also contain high levels of endotoxin. Accordingly, removal ofendotoxin from affinity-purified pMal-c and pMBot protein preparationswas attempted as follows.

[0601] Samples of pMal-c and pMBot protein were depyrogenated withpolymyxin to determine if the endotoxin could be easily removed. Thefollowing amount of protein was treated: 29 ml at 4.8 OD₂₈₀/ml forpMal-c and 19 mls at 1.44 OD₂₈₀/ml for pMBot. The protein samples weredialyzed extensively against PBS and mixed in a 50 ml tube (Falcon) with0.5 ml PBS-equilibrated polymyxin B (Affi-Prep Polymyxin, BioRad). Thesamples were allowed to mix by rotating the tubes overnight at 4° C. Thepolymyxin was pelleted by centrifugation for 30 min in a bench topcentrifuge at maximum speed (approximately 2000×g) and the supernatantwas removed. The recovered protein (in the supernatant) was quantifiedby OD₂₈₀, and the endotoxin activity was assayed by LAL. In both casesonly approximately ⅓ of the input protein was recovered and thepolymyxin-treated protein retained significant endotoxin contamination(approximately 7000 EU/mg of pMBot).

[0602] The depyrogenation experiment was repeated using an independentlypurified pMal-c protein preparation and similar results were obtained.From these studies it was concluded that significant levels of endotoxincopurifies with these MBP fusion proteins using the amylose resin.Furthermore, this endotoxin cannot be easily removed by polymyxintreatment.

[0603] These results suggest that the presence of the MBP sequences onthe fusion protein complicated the removal of endotoxin frompreparations of the pMBot protein.

[0604] b) Generation of C. botulinum C Fragment Protein Free of the MBP

[0605] It was demonstrated that the pMBot fusion protein could not beeasily purified from contaminating endotoxin in section a) above. Theability to produce a pyrogen-free (e.g., endotoxin-free) preparation ofsoluble botulinal C fragment protein free of the MBP tag was nextinvestigated. The pMBot expression construct was designed to facilitatepurification of the botulinal C fragment from the MBP tag by cleavage ofthe fusion protein by utilizing an engineered Factor Xa cleavage sitepresent between the MBP and the botulinal C fragment. The Factor Xacleavage was performed as follows.

[0606] Factor Xa (New England Biolabs) was added to the pMBot protein(using a 0.1-1.0% Factor Xa/pMBot protein ratio) in a variety of bufferconditions [e.g., PBS-NaCl (PBS containing 0.5 M NaCl), PBS-NaClcontaining 0.2% Tween 20, PBS, PBS containing 0.2% Tween 20, PBS-C (PBScontaining 2 mM CaCl₂), PBS-C containing either 0.1 or 0.5% Tween 20,PBS-C containing either 0.1 or 0.5% NP-40, PBS-C containing either 0.1or 0.5% Triton X-100, PBS-C containing 0.1% sodium deoxycholate, PBS-Ccontaining 0.1% SDS]. The Factor Xa digestions were incubated for 12-72hrs at room temperature.

[0607] The extent of cleavage was assessed by Western blot or Coomassieblue staining of proteins following electrophoresis on denaturingSDS-PAGE gels, as described in Example 22. Cleavage reactions (andcontrol samples of uncleaved pMBot protein) were centrifuged for 2 minin a microfuge to remove insoluble protein prior to loading the sampleson the gel. The Factor Xa treated samples were compared with uncleaved,uncentrifuged pMBot samples on the same gel. The results of thisanalysis is summarized below.

[0608] 1) Most (about 90%) pMBot protein could be removed bycentrifugation, even when uncleaved control samples were utilized. Thisindicated that the pMBot fusion protein was not fully soluble (i.e., itexists as a suspension rather than as a solution). [This result wasconsistent with the observation that most affinity-purified pMBotprotein precipitates after long term storage (>2 weeks) at 4° C.Additionally, the majority (i.e., 75%) of induced pMBot protein remainsin the pellet after sonication and clarification of the induced E. coli.Resuspension of these insoluble pellets in PBS followed by sonicationresults in partial solubilization of the insoluble pMBot protein in thepellets.]

[0609] 2) The portion of pMBot protein that is fully in solution (about10% of pMBot protein) is completely cleaved by Factor Xa, but thecleaved (released) botulinal C fragment is relatively insoluble suchthat only the cleaved MBP remains fully in solution.

[0610] 3) None of the above reaction conditions enhanced solubilitywithout also reducing effective cleavage. Conditions that effectivelysolubilized the cleaved botulinal C fragment were not identified.

[0611] 4) The use of 0.1% SDS in the buffer used for Factor Xa cleavageenhanced the solubility of the pMBot protein (all of pMBot protein wassoluble). However, the presence of the SDS prevented any cleavage of thefusion protein with Factor Xa.

[0612] 5) Analysis of pelleted protein from the cleavage reactionsindicated that both full length pMBot (i.e., uncleaved) and cleavedbotulinal C fragment protein precipitated during incubation.

[0613] These results demonstrate that purification of soluble botulinalC fragment protein after cleavage of the pMBot fusion protein iscomplicated by the insolubility of both the pMBot protein and thecleaved botulinal C fragment protein.

[0614] c) Expression of C. botulinum C Fragment Using Various ExpressionVectors

[0615] In order to determine if the solubility of the botulinal Cfragment was enhanced by expressing the C fragment protein as a nativeprotein, an N-terminal His-tagged protein or as a fusion withglutathione-S-transferase (GST), alternative expression plasmids wereconstructed. These expression constructs were generated utilizing themethodologies described in Example 22. FIG. 27 provides a schematicrepresentation of the vectors described below.

[0616] In FIG. 27, the following abbreviations are used. pP refers tothe pET23 vector. pHIS refers to the pETHisa vector. pBlue refers to thepBluescript vector. pM refers to the pMAL-c vector and pG refers to thepGEX3T vector (described in Example 11). The solid black lines representC. botulinum C fragment gene sequences; the solid black ovals representthe MBP; the hatched ovals represent GST; “HHHHH” represents thepoly-histidine tag. In FIG. 27, when the name for a restriction enzymeappears inside parenthesis, this indicates that the restriction site wasdestroyed during construction. An asterisk appearing with the name for arestriction enzyme indicates that this restriction site was recreated ata cloning junction.

[0617] i) Construction of pPBot

[0618] In order to express the C. botulinum C fragment as a native(i.e., non-fused) protein, the pPBot plasmid (shown schematically inFIG. 27) was constructed as follows. The C fragment sequences present inpAlterBot (Example 22) were removed by digestion of pAlterBot with NcoIand HindIII. The NcoI/HindIII C fragment insert was ligated to pETHisavector (described in Example 18b) which was digested with NcoI andHindIII. This ligation creates an expression construct in which theNcoI-encoded methionine of the botulinal C fragment is the initiatorcodon and directs expression of the native botulinal C fragment. Theligation products were used to transform competent BL21(DE3)pLysS cells(Novagen). Recombinant clones were identified by restriction mapping.

[0619] ii) Construction of pHisBot

[0620] In order to express the C. botulinum C fragment containing apoly-histidine tag at the amino-terminus of the recombinant protein, thepHisBot plasmid (shown schematically in FIG. 27) was constructed asfollows. The NcoI/HindIII botulinal C fragment insert from pAlterbot wasligated into the pETHisa vector which was digested with NheI andHindIII. The NcoI (on the C fragment insert) and NheI (on the pETHisavector) sites were filled in using the Klenow fragment prior toligation; these sites were then blunt end ligated (the NdeI site wasregenerated at the clone junction as predicted). The ligation productswere used to transform competent BL21(DE3)pLysS cells and recombinantclones were identified by restriction mapping.

[0621] The resulting pHisBot clone expresses the botulinal C fragmentprotein with a histidine-tagged N-terminal extension having thefollowing sequence: MetGlyH is His H is His H is His H is His H is HisSerSerGlyHisleGluGlyArgHisMetAla (SEQ ID NO:24); the amino acids encodedby the botulinal C fragment gene are underlined and the vector encodedamino acids are presented in plain type. The nucleotide sequence presentin the pETHisa vector which encodes the pHisBot fusion protein is listedin SEQ ID NO:25. The amino acid sequence of the pHisBot protein islisted in SEQ ID NO:26.

[0622] iii) Construction of pGBot

[0623] The botulinal C fragment protein was expressed as a fusion withthe glutathione-S-transferase protein by constructing the pGBot plasmid(shown schematically in FIG. 27). This expression construct was createdby cloning the NotI/SalI C fragment insert present in pBlueBot (Example22) into the pGEX3T vector which was digested with SmaI and XhoI. TheNotI site (present on the botulinal fragment) was made blunt prior toligation using the Klenow fragment. The ligation products were used totransform competent BL21 cells.

[0624] Each of the above expression constructs were tested byrestriction digestion to confirm the integrity of the constructs.

[0625] Large scale (1 liter) cultures of pPBot [BL21(DE3)pLysS host],pHisBot [BL21(DE3)pLysS host] and pGBot (BL21 host) were grown in 2×YTmedium and induced (using IPTG to 0.8-1.0 mM) for 3 hrs as described inExample 22. Total, soluble and insoluble protein preparations wereprepared from 1 ml aliquots of each large scale culture [Williams et al.(1994), supra] and analyzed by SDS-PAGE. No obvious induced band wasdetectable in the pPBot or pHisBot samples by Coomassie staining, whilea prominent insoluble band of the anticipated MW was detected in thepGBot sample. Soluble lysates of the pGBot large scale (resuspended inPBS) or pHisBot large scale [resuspended in Novagen 1× binding buffer (5mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9)] cultures wereprepared and used to affinity purify soluble affinity-tagged protein asfollows.

[0626] The pGBot lysate was affinity purified on a glutathione-agaroseresin (Pharmacia) exactly as described in Smith and Corcoran [CurrentProtocols in Molecular Biology, Supplement 28 (1994), pp.16.7.1-16.7.7]. The pHisBot protein was purified on the His-Bind resin(Novagen) utilizing the His-bind buffer kit (Novagen) exactly asdescribed by manufacturer.

[0627] Samples from the purification of both the pGBot and pHisBotproteins (including uninduced, induced, total, soluble, andaffinity-purified eluted protein) were resolved on SDS-PAGE gels.Following electrophoresis, proteins were analyzed by Coomassie stainingor by Western blot detection utilizing a chicken anti-C. botulinum TypeA toxoid antibody (as described in Example 22).

[0628] These studies showed that the pGBot protein was almost entirelyinsoluble under the utilized conditions, while the pHisBot protein wassoluble. Affinity purification of the pHisBot protein on this firstattempt was inefficient, both in terms of yield (most of theimmunoreactive botulinal protein did not bind to the His-bind resin) andpurity (the botulinal protein was estimated to comprise approximately20% of the total eluted protein).

[0629] d) Purification of Soluble C. botulinum C Fragment ProteinSubstantially Free of Endotoxin Contamination

[0630] The above studies showed that the pHisBot protein was expressedin E. coli as a soluble protein. However, the affinity purification ofthis protein on the His-bind resin was very inefficient. In order toimprove the affinity purification of the soluble pHisBot protein (interms of both yield and purity), an alternative poly-histidine bindingaffinity resin (Ni-NTA resin; Qiagen) was utilized. The Ni-NTA resin wasreported to have a superior binding affinity (K_(d)=1×10⁻¹³ at pH 8.0;Qiagen user manual) relative to the His-bind resin.

[0631] A soluble lysate (in Novagen 1× binding buffer) from an induced 1liter 2×YT culture was prepared as described above. Briefly, the cultureof pHisBot [B121(DE3)pLysS host] was grown at 37° C. to an OD₆₀₀ of 0.7in 1 liter of 2×YT medium containing 100 μg/ml ampicillin, 34 μg/mlchloramphenicol and 0.2% glucose. Protein expression was induced by theaddition of IPTG to 1 mM. Three hours after the addition of the IPTG,the cells were cooled for 15 min in a ice water bath and thencentrifuged 10 min at 5000 rpm in a JA10 rotor (Beckman) at 4° C. Thepellets were resuspended in a total volume of 40 mls Novagen 1× bindingbuffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9), transferredto two 35 ml Oakridge tubes and frozen at −70° C. for at least 1 hr. Thetubes were thawed and the cells were lysed by sonication (4×20 secondbursts using a Branson Sonifier 450 with a power setting of 6-7) on ice.The suspension was clarified by centrifugation for 20 min at 9,000 rpm(10,000×g) in a JA-17 rotor (Beckman).

[0632] The soluble lysate was brought to 0.1% NP40 and then was batchabsorbed to 7 ml of a 1:1 slurry of Ni-NTA resin:binding buffer bystirring for 1 hr at 4° C. The slurry was poured into a column having aninternal diameter of 1 or 2.5 cm (BioRad). The column was then washedsequentially with 15 mls of Novagen 1× binding buffer containing 0.1%NP40, 15 ml of Novagen 1× binding buffer, 15 ml wash buffer (60 mMimidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) and 15 ml NaHPO₄ washbuffer (50 mM NaHPO₄, pH 7.0, 0.3 M NaCl, 10% glycerol). The boundprotein was eluted by protonation of the resin using elution buffer (50mM NaHPO₄, pH 4.0, 0.3 M NaCl, 10% glycerol). The eluted protein wasstored at 4° C.

[0633] Samples of total, soluble and eluted protein were resolved bySDS-PAGE. Protein samples were prepared for electrophoresis as describedin Example 22b. Duplicate gels were stained with Coomassie blue tovisualize the resolved proteins and C. botulinum type A toxin-reactiveprotein was detected by Western blot analysis as described in Example22b. A representative Coomassie stained gel is shown in FIG. 28. In FIG.28, the following samples were loaded on the 12.5% acrylamide gel. Lanes1-4 contain respectively total protein, soluble protein, soluble proteinpresent in the flow-through of the Ni-NTA column and affinity-purifiedpHisBot protein (i.e., protein released from the Ni-NTA resin byprotonation). Lane 5 contains high molecular weight protein markers(BioRad).

[0634] The purification of pHisBot protein resulted in a yield of 7 mgof affinity purified protein from a 1 liter starting culture ofBL21(DE3)pLysS cells harboring the pHisBot plasmid. The yield ofpurified pHisBot protein represented approximately 0.4% of the totalsoluble protein in the induced culture. Analysis of the purified pHisBotprotein by SDS-PAGE revealed that at least 90-95% of the protein waspresent as a single band (FIG. 28) of the predicted MW (50 kD). This 50kD protein band was immunoreactive with anti-C. botulinum type A toxinantibodies. The extinction coefficient of the protein preparation wasdetermined to be 1.4 (using the Pierce BCA assay) or 1.45 (using theLowry assay) OD₂₈₀ per 1 mg/ml solution.

[0635] Samples of pH neutralized eluted pHisBot protein were resolved ona KB 803 HPLC column (Shodex). Although His-tagged proteins are retainedby this sizing column (perhaps due to the inherent metal binding abilityof the proteins), the relative mobility of the pHisBot protein wasconsistent with that expected for a non-aggregated protein in solution.Most of the induced pHisBot protein was determined to be soluble underthe growth and solubilization conditions utilized above (i.e., greaterthan 90% of the pHisBot protein was found to be soluble as judged bycomparison of the levels of pHisBot protein seen in total and solubleprotein samples prepared from BL21(DE3)pLysS cells containing thepHisBot plasmid). SDS-PAGE analysis of samples obtained aftercentrifugation, extended storage at −20° C., and at least 2 cycles offreezing and thawing detected no protein loss (due to precipitation),indicating that the pHisBot protein is soluble in the elution buffer(i.e., 50 mM NaHPO₄, pH 4.0, 0.3 M NaCl, 10% glycerol).

[0636] Determination of endotoxin contamination in the affinity purifiedpHisBot preparation (after pH neutralization) using the LAL assay(Associates of Cape Cod) detected no significant endotoxincontamination. The assay was performed using the endpoint chromogenicmethod (without diazo-coupling) according to the manufacturer'sinstructions. This method can detect concentrations of endotoxin greaterthan or equal to 0.03 EU/ml (EU refers to endotoxin units). The LALassay was run using 0.5 ml of a solution comprising 0.5 mg. pHisBotprotein in 50 mM NaHPO₄, pH 7.0, 0.3 M NaCl, 10% glycerol; 30-60 EU weredetected in the 0.5 ml sample. Therefore, the affinity purified pHisBotpreparation contains 60-120 EU/mg of protein. FDA Guidelines for theadministration of parenteral drugs require that a composition to beadministered to a human contain less than 5 EU/kg body weight (Theaverage human body weight is 70 kg; therefore up to 349 EU units can bedelivered in a parental dose.). Because very small amount of protein areadministered in a vaccine preparation (generally in the range of 10-500μg of protein), administration of affinity purified pHisBot containing60-120 EU/mg protein would result in delivery of only a small percentageof the permissible endotoxin load. For example, administration of 10-500μg of purified pHisBot to a 70 kg human, where the protein preparationcontains 60 EU/mg protein, results in the introduction of only 0.6 to 30EU [i.e., 0.2 to 8.6% of the maximum allowable endotoxin burden perparenteral dose (less than 5 EU/kg body weight)].

[0637] The above results demonstrate that endotoxin (LPS) does notcopurify with the pHisBot protein using the above purification scheme.Preparations of recombinantly produced pHisBot protein containing lowerlevels of endotoxin (less than or equal to 2 EU/mg recombinant protein)may be produced by washing the Ni-NTA column with wash buffer until theOD₂₈₀ returns to baseline levels (i.e., until no more UV-absorbingmaterial comes off of the column).

[0638] The above results illustrate a method for the production andpurification of soluble, botulinal C fragment protein substantially freeof endotoxin.

Example 25 Optimization of the Expression and Purification of pHisBotProtein

[0639] The results shown in Example 24d demonstrated that the pHisBotprotein is an excellent candidate for use as a vaccine as it could beproduced as a soluble protein in E. coli and could be purified free ofpyrogen activity. In order to optimize the expression and purificationof the pHisBot protein, a variety of growth and purification conditionswere tested.

[0640] a) Growth Parameters

[0641] i) Host Strains

[0642] The influence of the host strain utilized upon the production ofsoluble pHisBot protein was investigated. A large scale purification ofpHisBot was performed [as described in Example 24d above] using theBL21(DE3) host (Novagen) rather than the BL21(DE3)pLysS host. Thedeletion of the pLysS plasmid in the BL21(DE3) host yielded higherlevels of expression due to de-repression of the plasmid's T7-lacpromoter. However, the yield of affinity-purified soluble recombinantprotein was very low (approximately 600 μg/liter culture) when purifiedunder conditions identical to those described in Example 24d above. Thisresult was due to the fact that expression in the BL21(DE3) host yieldedvery high level expression of the pHisBot protein as insoluble inclusionbodies as shown by SDS-PAGE analysis of protein prepared from inducedBL21(DE3) cultures (FIG. 29, lanes 1-7, described below). These resultsdemonstrate that the pHisBot protein is not inherently toxic to E. colicells and can be expressed to high levels using the appropriatepromoter/host combination.

[0643]FIG. 29 shows a Coomassie blue stained SDS-PAGE gel (12.5%acrylamide) onto which extracts prepared from BL21(DE3) cells containingthe pHisBot plasmid were loaded. Each lane was loaded with 2.5 μlprotein sample mixed with 2.5 μl of 2×SDS sample buffer. The sampleswere handled as described in Example 22b. The following samples wereapplied to the gel. Lanes 1-7 contain protein isolated from theBL21(DE3) host. Lanes 8-14 contain proteins isolated from theBL21(DE3)pLysS host. Total protein was loaded in lanes 1, 2, 4, 6, 8, 10and 12. Soluble protein was loaded in Lanes 3, 5, 7, 9, 11 and 13. Lane1 contains protein from uninduced host cells. Lanes 2-13 contain proteinfrom host cells induced for 3 hours. IPTG was added to a finalconcentration of 0.1 mM (Lanes 6-7), 0.3 mM (Lanes 4-5) or 1.0 mM (Lanes2, 3, 8-13). The cultures were grown in LB broth (Lanes 8-9), 2×YT broth(Lanes 10-11) or terrific broth (Lanes 1-7, 12-13). The pHisBot proteinseen in Lanes 3, 5 and 7 is insoluble protein which spilled over fromLanes 2, 4 and 6, respectively. High molecular weight protein markers(BioRad) were loaded in Lane 14.

[0644] A variety of expression conditions were tested to determine ifthe BL21(DE3) host could be utilized to express soluble pHisBot proteinat suitably high levels (i.e., about 10 mg/ml). The conditions alteredwere temperature (growth at 37 or 30° C.), culture medium (2×YT, LB orTerrific broth) and inducer levels (0.1, 0.3 or 1.0 mM IPTG). Allcombinations of these variables were tested and the induction levels andsolubility was then assessed by SDS-PAGE analysis of total and solubleextracts [prepared from 1 ml samples as described in Williams et al.,(1994), supra].

[0645] All cultures were grown in 15 ml tubes (Falcon #2057). Allculture medium was prewarmed overnight at the appropriate temperatureand were supplemented with 100 μg/ml ampicillin and 0.2% glucose.Terrific broth contains 12 g/l bacto-tryptone, 24 g/l bacto-yeastextract and 100 ml/l of a solution comprising 0.17 M KH₂PO₄, 0.72 MK₂HPO₄. Cultures were grown in a incubator on a rotating wheel (toensure aeration) to an OD₆₀₀ of approximately 0.4, and induced by theaddition of IPTG. In all cases, high level expression of insolublepHisBot protein was observed, regardless of temperature, medium orinducer concentration.

[0646] The effect of varying the concentration of IPTG upon 2×YTcultures grown at 23° C. was then investigated. IPTG was added to afinal concentration of either 1 mM, 0.1 mM, 0.05 mM or 0.01 mM. At thistemperature, similar levels of pHis Bot protein was induced in thepresence of either 1 or 0.1 mM IPTG; these levels of expression waslower than that observed at higher temperatures. Induced protein levelswere reduced at 0.05 mM IPTG and absent at 0.01 mM IPTG (relative to 1.0and 0.1 mM IPTG inductions at 23° C.). However, no conditions wereobserved in which the induced pHisBot protein was soluble in this host.Thus, although expression levels are superior in the BL21(DE3) host (ascompared to the BL21(DE3)pLysS host), conditions that facilitate theproduction of soluble protein in this host could not be identified.

[0647] These results demonstrate that production of soluble pHisBotprotein was achieved using the BL21(DE3)pLysS host in conjunction withthe T7-lac promoter.

[0648] ii) Effect of Varying Temperature, Medium and IPTG Concentrationand Length of Induction

[0649] The effect growing the host cells in various mediums upon theexpression of recombinant botulinal protein from the pHisBot expressionconstruct [in the BL21(DE3)pLysS host] was investigated. BL21(DE3)pLysScells containing the pHisBot plasmid were grown in either LB, 2×YT orTerrific broth at 37° C. The cells were induced using 1 mM IPTG for a 3hr induction period. Expression of pHisBot protein was found to be thehighest when the cells were grown in 2×YT broth (see FIG. 29, lanes8-13).

[0650] The cells were then grown at 30° C. in 2×YT broth and theconcentration of IPTG was varied from 1.0, 0.3 or 0.1 mM and the lengthof induction was either 3 or 5 hours. Expression of pHisBot protein wassimilar at all 3 inducer concentrations utilized and the levels ofinduced protein were higher after a 5 hr induction as compared to a 3 hrinduction.

[0651] Using the conditions found to be optimal for the expression ofpHisBot protein, a large scale culture was grown in order to providesufficient material for a large scale purification of the pHisBotprotein. Three 1 liter cultures were grown in 2×YT medium containing 100μg/ml ampicillin, 34 μg/ml chloramphenicol and 0.2% glucose. Thecultures were grown at 30° C. and were induced with 1.0 mM IPTG for a 5hr period. The cultures were harvested and a soluble lysate wereprepared as described in Example 18. A large scale purification wasperformed as described in Example 24d with the exception that except thesoluble lysate was batch absorbed for 3 hours rather than for 1 hour.The final yield was 13 mg pHisBot protein/liter culture. The pHisBotprotein represented 0.75% of the total soluble protein.

[0652] The above results demonstrate growth conditions under whichsoluble pHisBot protein is produced (i.e., use of the BL21(DE3)pLysShost, 2×YT medium, 30° C., 1.0 mM IPTG for 5 hours).

[0653] b) Optimization of Purification Parameters

[0654] For optimization of purification conditions, large scale cultures(3×1 liter) were grown at 30° C. and induced with 1 mM IPTG for 5 hoursas described above. The cultures were pooled, distributed to centrifugebottles, cooled and pelleted as described in Example 24d. The cellpellets were frozen at −70° C. until used. Each cell pellet represented⅓ of a liter starting culture and individual bottles were utilized foreach optimization experiment described below. This standardized theinput bacteria used for each experiment, such that the yields ofaffinity purified pHisBot protein could be compared between differentoptimization experiments.

[0655] i) Binding Specificity (pH Protonation)

[0656] A lysate of pHisBot culture was prepared in PBS (pH 8.0) andapplied to a 3 ml Ni-NTA column equilibrated in PBS (pH 8.0) using aflow rate of 0.2 ml/min (3-4 column volumes/hr) using an Econochromatography system (BioRad). The column was washed with PBS (pH 8.0)until the absorbance (OD₂₈₀) of the elute was at baseline levels. Theflow rate was then increased to 2 ml/min and the column was equilibratedin PBS (pH 7.0). A pH gradient (pH 7.0 to 4.0 in PBS) was applied inorder to elute the bound pHisBot protein from the column. Fractions werecollected and aliquots were resolved on SDS-PAGE gels. The PAGE gelswere subjected to Western blotting and the pHisBot protein was detectedusing a chicken anti-C. botulinum Type A toxoid antibody as described inExample 22.

[0657] From the Western blot analysis it was determined that the pHisBotprotein begins to elute from the Ni-NTA column at pH 6.0. This isconsistent with the predicted elution of a His-tagged protein monomer atpH 5.9.

[0658] These results demonstrate that the pH at which the pHisBotprotein is protonated (released) from Ni-NTA resin in PBS buffer is pH6.0.

[0659] ii) Binding Specificity (Imidazole Competition)

[0660] In order to define purification conditions under which the nativeE. coli proteins could be removed from the Ni-NTA column while leavingthe pHisBot protein bound to the column, the fellowing experiment wasperformed. A lysate of pHisBot culture was prepared in 50 mM NaHPO₄, 0.5M NaCl, 8 mM imidazole (pH 7.0). This lysate was applied to a 3 mlNi-NTA column equilibrated in 50 mM NaHPO₄, 0.5 M NaCl (pH 7.0) using anEcono chromatography system (BioRad). A flow rate of 0.2 ml/min (3-4column volumes/hr) was utilized. The column was washed with 50 mMNaHPO₄, 0.5 M NaCl (pH 7.0) until the absorbance of the elute returnedto baseline. The flow rate was then increased to 2 ml/min.

[0661] The column was eluted using an imidazole step gradient [in 50 mMNaHPO₄, 0.5 M NaCl (pH 7.0)]. Elution steps were 20 mM, 40 mM, 60 mM, 80mM, 100 mM, 200 mM, 1.0 M imidazole, followed by a wash using 0.1 mMEDTA (to strip the nickel from the column and remove any remainingprotein). In each step, the wash was continued until the OD₂₈₀ returnedto baseline. Fractions were resolved on SDS-PAGE gels, Western blotted,and pHisBot protein detected using a chicken anti-C. botulinum Type Atoxoid antibody as described in Example 22. Duplicate gels were stainedwith Coomassie blue to detect eluted protein in each fraction.

[0662] The results of the PAGE analysis showed that most of thenon-specifically binding bacterial protein was removed by the 20 mMimidiazole wash, with the remaining bacterial proteins being removed inthe 40 and 60 mM imidazole washes. The pHisBot protein began to elute at100 mM imidazole and was quantitatively eluted in 200 mM imidazole.

[0663] These results precisely defined the window of imidazole washstringency that optimally removes E. coli proteins from the column whilespecifically retaining the pHisBot protein in this buffer. These resultsprovided conditions under which the pHisBot protein can be purified freeof contaminating host proteins.

[0664] iii) Purification Buffers and Optimized Purification Protocols

[0665] A variety of purification parameters were tested during thedevelopment of an optimized protocol for batch purification of solublepHisBot protein. The results of these analyses are summarized below.

[0666] Batch purifications were performed (as described in Example 24d)using several buffers to determine if alternative buffers could beutilized for binding of the pHisBot protein to the Ni-NTA column. It wasdetermined that quantitative binding of pHisBot protein to the Ni-NTAresin was achieved in either Tris-HCl (pH 7.9) or NaHPO₄ (pH 8.0)buffers. Binding of the pHisBot protein in NaHPO₄ buffer was notinhibited using 5 mM, 8 mM or 60 mM imidazole. Quantitative elution ofbound pHisBot protein was obtained in buffers containing 50 mM NaHPO₄,0.3 M NaCl (pH 3.5-4.0), with or without 10% glycerol. However,quantitation of soluble affinity purified pHisBot protein before andafter a freeze thaw (following several weeks storage of the affinitypurified elute at −20° C.) revealed that 94% of the protein wasrecovered using the glycerol-containing buffer, but only 68% of theprotein was recovered when the buffer lacking glycerol was employed.This demonstrates that glycerol enhanced the solubility of the pHisBotprotein in this low pH buffer when the eluted protein was stored atfreezing temperatures (e.g., −20° C.). Neutralization of pH by additionof NaH₂PO₄ buffer did not result in obvious protein precipitation.

[0667] It was determined that quantitative binding of pHisBot proteinusing the batch format occurred after 3 hrs (FIG. 30), but not after 1hr of binding at 4° C. (the resin was stirred during binding). FIG. 30depicts a Coomaisse blue stained SDS-PAGE gel (7.5% acrylamide)containing samples of proteins isolated during the purification ofpHisBot protein from lysate prepared from the BL21(DE3)pLysS host. Eachlane was loaded with 5 μl of protein sample mixed with 5 μl of 2× samplebuffer and processed as described in Example 22b. Lane 1 contains highmolecular weight protein markers (BioRad). Lanes 2 and 3 contain proteineluted from the Ni-NTA resin. Lane 4 contains soluble protein after a 3hr batch incubation with the Ni-NTA resin. Lanes 5 and 6 contain solubleand total protein, respectively. FIG. 30 demonstrates that the pHisBotprotein is completely soluble [compare Lanes 5 and 6 which show that asimilar amount of the 50 kD pHisBot protein is seen in both; if asubstantial amount (greater than 20%) of the pHisBot protein werepartially insoluble in the host cell, more pHisBot protein would be seenin lane 6 (total protein) as compared to lane 5 (soluble protein)]. FIG.30 also demonstrates that the pHisBot protein is completely removed fromthe lysate after batch absorption with the Ni-NTA resin for 3 hours(compare Lanes 4 and 5).

[0668] The reported high affinity interaction of the Ni-NTA resin withHis-tagged proteins (K_(d)=1×10⁻¹³ at pH 8.0) suggested that it shouldbe possible to manipulate the resin-protein complexes withoutsignificant release of the bound protein. Indeed, it was determined thatafter the recombinant protein was bound to the Ni-NTA resin, theresin-pHisBot protein complex was highly stable and remained boundfollowing repeated rounds of centrifugation of the resin for 2 min at1600×g. When this centrifugation step was performed in a 50 ml tube(Falcon), a tight resin pellet formed. This allowed the removal of spentsoluble lysate by pouring off the supernatant followed by resuspensionof the pellet in wash buffer. Further washes can be performed bycentrifugation. The ability to perform additional washes permits thedevelopment of protocols for batch absorption of large volumes of lysatewith removal of the lysate being performed simply by centrifugationfollowing binding of the recombinant protein to the resin.

[0669] A simplified, integrated purification protocol was developed asfollows. A soluble lysate was made by resuspending the induced cellpellet in binding buffer [50 mM NaHPO₄, 0.5 M NaCl, 60 mM imidazole (pH8.0)], sonicating 4×20 sec and centrifuging for 20 min at 10,000×g.NP-40 was added to 0.1% and Ni-NTA resin (equilibrated in bindingbuffer) was added. Eight milliliters of a 1:1 slurry (resin:bindingbuffer) was used per liter of starting culture. The mixture was stirredfor 3 hrs at 4° C. The slurry was poured into a column having a 1 cminternal diameter (BioRad), washed with binding buffer containing 0.1%NP40, then binding buffer until baseline was established (these stepsmay alternatively be performed by centrifugation of the resin,resuspension in binding buffer containing NP40 followed bycentrifugation and resuspension in binding buffer). Imidazole wasremoved by washing the resin with 50 mM NaHPO₄, 0.3M NaCl (pH 7.0).Protein bound to the resin was eluted using the same buffer (50 mMNaHPO₄, 0.3M NaCl) having a reduced pH (pH 3.5-4.0).

[0670] A pilot purification was performed following this protocol andyielded 18 mg/liter affinity-purified pHisBot. The pHisBot protein wasgreater than 90% pure as estimated by Coomassie staining of an SDS-PAGEgel. This represents the highest observed yield of solubleaffinity-purified pHisBot protein and this protocol eliminates the needfor separate imidazole-containing binding and wash buffers. In additionto providing a simplified and efficient protocol for the affinitypurification of recombinant pHisBot protein, the above results provide avariety of purification conditions under which pHisBot protein can beisolated.

Example 26 The pHisBot Protein is an Effective Immunogen

[0671] In Example 23 it was demonstrated that neutralizing antibodiesare generated in mouse serum after nasal immunization with the pMBotprotein. However, the pMBot protein was found to copurify withsignificant amounts of endotoxin which could not be easily removed. ThepHisBot protein, in contrast, could be isolated free of significantendotoxin contamination making pHisBot a superior candidate for vaccineproduction. To further assess the suitability of pHisBot as a vaccine,the immunogenicity of the pHisBot protein was determined and acomparison of the relative immunogenicity of pMBot and pHisBot proteinsin mice was performed as follows.

[0672] Two groups of eight BALBc mice were immunized with either pMBotprotein or pHisBot protein using Gerbu GMDP adjuvant (CC Biotech). pMBotprotein (in PBS containing 10 mM maltose) or pHisBot protein (in 50mMNaHPO₄, 0.3 M NaCl, 10% glycerol, pH 4.0) was mixed with Gerbuadjuvant and used to immunize mice. Each mouse received an IP injectionof 100 μl antigen/adjuvant mix (50 pg antigen plus 1 μg adjuvant) on day0. Mice were boosted as described above with the exception that theroute of administration was IM on day 14 and 28. The mice were bled onday 77 and anti-C. botulinum Type A toxoid titers were determined usingserum collected from individual mice in each group (as described inExample 23). The results are shown in Table 41. TABLE 41 Anti - C.botulinum Type A Toxoid Serum IgG Titers In Individual Mice ImmunizedWith pMBot or pHisBot Protein Preimmune¹ pMBot² pHisBot² Sample DilutionSample Dilution Sample Dilution Mouse # 1:50 1:250 1:1250 1:6250 1:501:250 1:1250 1:6250 1:50 1:250 1:1250 1:620 1 0.678 0.190 0.055 0.0071.574 0.799 0.320 0.093 2 1.161 0.931 0.254 0.075 1.513 0.829 0.4090.134 3 1.364 0.458 0.195 0.041 1.596 1.028 0.453 0.122 4 1.622 1.1890.334 0.067 1.552 0.840 0.348 0.090 5 1.612 1.030 0.289 0.067 1.6291.580 0.895 0.233 6 0.913 0.242 0.069 0.013 1.485 0.952 0.477 0.145 70.910 0.235 0.058 0.014 1.524 0.725 0.269 0.069 8 0.747 0.234 0.0580.014 1.274 0.427 0.116 0.029 Mean 0.048 0.021 0.011 0.002 1.133 0.5640.164 0.037 1.518 0.896 0.411 0.114 Titer

[0673] The results shown above in Table 41 demonstrate that both thepMBot and pHisBot proteins are immunogenic in mice as 100% of the mice(8/8) in each group seroconverted from non-immune to immune status. Theresults also show that the average titer of anti-C. botulinum Type Atoxoid IgG is 2-3 fold higher after immunization with the pHisBotprotein relative to immunization with the pMBot protein. This suggeststhat the pHisBot protein may be a superior immunogen to the pMBotprotein.

Example 27 Immunization with the Recombinant pHisBot Protein GeneratesNeutralizing Antibodies

[0674] The results shown in Example 26 demonstrated that both thepHisBot and pMBot proteins were capable of inducing high titers ofanti-C. botulinum type A toxoid-reactive antibodies in immunized hosts.The ability of the immune sera from mice immunized with either thepHisBot or pMBot proteins to neutralize C. botulinum type A toxoid invivo was determined using the mouse neutralization assay described inExample 23b.

[0675] The two groups of eight BALBc mice immunized with either pMBotprotein or pHisBot protein in Example 26 were boosted again one weekafter the bleeding on day 77. The boost was performed by mixing pMBotprotein (in PBS containing 10 mM maltose) or pHisBot protein (in 50 mMNaHPO₄, 0.3 M NaCl, 10% glycerol, pH 4.0) with Gerbu adjuvant asdescribed in Example 26. Each mouse received an IP injection of 100 μlantigen/adjuvant mix (50 μg antigen plus 1 μg adjuvant). The mice werebled 6 days after this boost and the serum from mice within a group waspooled. Serum from preimmune mice was also collected (this serum is thesame serum described in the footnote to Table 41).

[0676] The presence of neutralizing antibodies in the pooled orpreimmune serum was detected by challenging mice with 5 LD₅₀ units oftype A toxin mixed with 100 μl of pooled serum. The challenge wasperformed by mixing (per mouse to be injected) 100 μl of serum from eachpool with 100 μl of purified type A toxin standard (50 LD₅₀/ml preparedas described in Example 23b) and 500 μl of gel-phosphate. The mixtureswere incubated for 30 min at room temperature with occasional mixing.Each of four mice were injected IP with the mixtures (0.7 ml/mouse). Themice were observed for signs of botulism for 72 hours. Mice receivingtoxin mixed with serum from mice immunized with either the pHisBot orpMBot proteins showed no signs of botulism intoxication. In contrast,mice receiving preimmune serum died in less than 24 hours.

[0677] These results demonstrate that antibodies capable of neutralizingC. botulinum type A toxin are induced when either of the recombinant C.botulinum C fragment proteins pHisBot or pMBot are used as immunogens.

Example 28 Cloning and Expression of the C Fragment of C. botulinumSerotype A Toxin in E. coli Utilizing a Native Gene Fragment

[0678] In Example 22 above, a synthetic gene was used to express the Cfragment of C. botulinum serotype A toxin in E. coli. The synthetic genereplaced non-preferred (i.e., rare) codons present in the C fragmentgene with codons which are preferred by E. coli. The synthetic gene wasgenerated because it was been reported that genes which have a high A/Tcontent (such as most clostridial genes) creates expression difficultiesin E. coli and yeast. Furthermore, LaPenotiere et al. suggested thatproblems encountered with the stability (non-fusion constructs) andsolubility (MBP fusion constructs) of the C fragment of C. botulinumserotype A toxin when expressed in E. coli was most likely due to theextreme A/T richness of the native C. botulinum serotype. A toxin genesequences (LaPenotiere, et al., supra).

[0679] In this example, it was demonstrated that successful expressionof the C fragment of C. botulinum type A toxin gene in E. coli does notrequire the elimination of rare codons (i.e., there is no need to use asynthetic gene). This example involved a) the cloning of the native Cfragment of the C. botulinum serotype A toxin gene and construction ofan expression vector and b) a comparison of the expression andpurification yields of C. botulinum serotype A C fragments derived fromnative and synthetic expression vectors.

[0680] a) Cloning of the Native C Fragment of the C. botulinum SerotypeA Toxin Gene and Construction of an Expression Vector

[0681] The serotype A toxin gene was cloned from C. botulinum genomicDNA using PCR amplification. The following primer pair was employed:5′-CGCCATGGCTAG ATTATTATCTACATTTAC-3′ (5′ primer, NcoI site underlined;SEQ ID NO:29) and 5′-GCAAGCTTCTTGACAGACTCATGTAG-3′ (3′ primer, HindIIIsite underlined; SEQ ID NO:30). C. botulinum type A strain was obtainedfrom the American Type Culture Collection (ATCC#19397) and grown underanaerobic conditions in Terrific broth medium. High molecular-weight C.botulinum DNA was isolated as described in Example 11. The integrity andyield of genomic DNA was assessed by comparison with a serial dilutionof uncut lambda DNA after electrophoresis on an agarose gel.

[0682] The gene fragment was cloned by PCR utilizing a proofreadingthermostable DNA polymerase (native Pfu polymerase). PCR amplificationwas performed using the above primer pair in a 50 μl reaction containing10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 200 μM each dNTP, 0.2μM each primer, and 50 ng C. botulinum genomic DNA. Reactions wereoverlaid with 100 μl mineral oil, heated to 94° C. 4 min, 0.5 μl nativePfu polymerase (Stratagene) was added, and thirty cycles comprising 94°C. for 1 min, 50° C. for 2 min, 72° C. for 2 min were carried outfollowed by 10 min at 72° C. An aliquot (10 μl) of the reaction mixturewas resolved on an agarose gel and the amplified native C fragment genewas gel purified using the Prep-A-Gene kit (BioRad) and ligated topCRScript vector DNA (Stratagene). Recombinant clones were isolated andconfirmed by restriction digestion, using standard recombinant molecularbiology techniques [Sambrook et al. (1989), supra]. In addition, thesequence of approximately 300 bases located at the 5′ end of the Cfragment coding region were obtained using standard DNA sequencingmethods. The sequence obtained was identical to that of the publishedsequence.

[0683] An expression vector containing the native C. botulinum serotypeA C fragment gene was created by ligation of the NcoI-HindIII fragmentcontaining the C fragment gene from the pCRScript clone to NheI-HindIIIrestricted pETHisa vector (Example 18b). The NcoI and NheI sites werefilled in using the Klenow enzyme prior to ligation; these sites werethus blunt-end ligated together. The resulting construct was termedpHisBotA (native). pHisBotA (native) expresses the C. botulinum serotypeA C fragment with a his-tagged N terminal extension which has thefollowing sequence:MetGlyHisHisHisHisHisHisHisHisHisHisSerSerGlyHisIleG/uGlyArgHisMetAla(SEQ ID NO:24), where the underlining represents amino acids encoded bythe C. botulinum C fragment gene (this N terminal extension contains therecognition site for Factor Xa protease, shown in italics, which can beemployed to removed the polyhistdine tract from the N-terminus of thefusion protein). The pHisBot (native) construct expresses the identicalprotein as the pHisBot construct (Ex. 24c; herein after the pHisBotA)which contains the synthetic gene.

[0684] The predicted DNA sequence encoding the native C. botulinumserotype A C fragment gene contained within pHisBotA (native) is listedin SEQ ID NO:31 (the start of translation (ATG) is located atnucleotides 108-110 and the stop of translation (TAA) is located atnucleotides 1494-1496 in SEQ ID NO:31] and the corresponding amino acidsequence is listed in SEQ ID NO:26 (i.e., the same amino acid sequenceas that produced by pHisBotA containing synthetic gene sequences).

[0685] b) Comparison of the Expression and Purification Yields of C.botulinum Serotype A C Fragments Derived From Native and SyntheticExpression Vectors

[0686] Recombinant plasmids containing either the native or thesynthetic C. botulinum serotype A C fragment genes were transformed intoE. coli strain B121(DE3) pLysS and protein expression was induced in 1liter shaker flask cultures. Total protein extracts were isolated,resolved on SDS-PAGE gels and C. botulinum C fragment protein wasidentified by Western analysis utilizing a chicken anti-C. botulinumserotype A toxoid antiserum as described in Example 22.

[0687] Briefly, 1 liter (2XYT+100 μg/ml ampicillin and 34 μg/mlchloramphenicol) cultures of bacteria harboring either the pHisBotA(synthetic) or pHisBotA (native) plasmids in the B121(DE3) pLysS strainwere induced to express recombinant protein by addition of IPTG to 1 mM.Cultures were grown at 30-32° C., IPTG was added when the cell densityreached an OD₆₀₀ 0.5-1.0 and the induced protein was allowed toaccumulate for 3-4 hrs after induction.

[0688] The cells were cooled for 15 min in a ice water bath and thencentrifuged for 10 min at 5000 rpm in a JA10 rotor (Beckman) at 4° C.The cell pellets were resuspended in a total volume of 40 mls 1× bindingbuffer (40 mM imidazole, 0.5 M NaCl, 50 mM NaPO₄, pH 8.0), transferredto two 50 ml Oakridge tubes and frozen at −70° C. for at least 1 hr. Thetubes were then thawed and the cells were lysed by sonication (usingfour successive 20 second bursts) on ice. The suspension was clarifiedby centrifugation 20-30 min at 9,000 rpm (10,000 g) in a JA-17 rotor.The soluble lysate was batch absorbed to 7 ml of a 1:1 slurry of NiNTAresin:binding buffer by stirring 2-4 hr at 4° C. The slurry wascentrifuged for 1 min at 500 g in 50 ml tube (Falcon), resuspended in 5mls binding buffer and poured into a 2.5 cm diameter column (BioRad).The column was attached to a UV monitor (ISCO) and the column was washedwith binding buffer until a baseline was established. Imidazole wasremoved by washing with 50 mM NAPO₄, 0.3 M NaCl, 10% glycerol, pH 7.0and bound protein was eluted using 50 mM NaPO₄, 0.3 M NaCl, 10%glycerol, pH 3.5-4.0.

[0689] The eluted proteins were stored at 4° C. Samples of total,soluble, and eluted proteins were resolved by SDS-PAGE. Protein sampleswere prepared for electrophoresis by mixing 1 μl total (T) or soluble(S) protein with 4 μl PBS and 5 μl 2×SDS-PAGE sample buffer, or 5 μleluted (E) protein and 5 μl 2×SDS-PAGE sample buffer. The samples wereheated to 95° C. for 5 min, then cooled and 5 or 10 μs were loaded on12.5% SDS-PAGE gels. Broad range molecular weight protein markers(BioRad) were also loaded to allow the MW of the identified fusionproteins to be estimated. After electrophoresis, protein was detectedeither generally by staining gels with Coomassie blue, or specifically,by blotting to nitrocellulose for Western blot detection of specificimmunoreactive protein.

[0690] For Western blot analysis, the gels were blotted, and proteintransfer was confirmed by Ponceau S staining as described in Example 22.After blocking the blots for 1 hr at room temperature in blocking buffer(PBST and 5% milk), 10 ml of a {fraction (1/500)} dilution of an anti-C.botulinum toxin A IgY PEG prep (Ex. 3) in blocking buffer was added andthe blots were incubated for an additional hour at room temperature. Theblots were washed and developed using a rabbit anti-chicken alkalinephosphatase conjugate (Boehringer Mannheim) as the secondary antibody asdescribed in Ex. 22. This analysis detected C. botulinum toxinA-reactive proteins in the pHisBotA (native and synthetic) proteinsamples (corresponding to the predicted full length proteins identifiedby Coomassie staining).

[0691] A gel containing proteins expressed from the pHisBot and pHisBot(native) constructs during various stages of purification and stainedwith Coomassie blue is shown in FIG. 31. In FIG. 31, lanes 1-4 and 9contain proteins expressed by the pHisBotA construct (i.e., thesynthetic gene) and lanes 5-8 contain proteins expressed by the pHisBotA(native) construct. Lanes 1 and 5 contain total protein extracts; lanes2 and 6 contain soluble protein extracts; lanes 3 and 7 contain proteinswhich flowed through the NiNTA columns; lanes 4, 8 and 9 contain proteineluted from the NiNTA columns and lane 10 contains molecular weightmarkers.

[0692] The above purification resulted in a yield of 3 mg (native gene)or 11 mg (synthetic gene) of affinity purified protein from a 1 literstarting culture, of which at least 90-95% of the protein was a singleband of the predicted MW (50 kd) and immunoreactivity for recombinant C.botulinum serotype A C fragment protein. Other than the level ofexpression, no difference was observed between the native and thesynthetic gene expression systems.

[0693] These results demonstrate that soluble C. botulinum serotype A Cfragment protein can be expressed in E. coli and purified utilizingeither native or synthetic gene sequences.

Example 29 Generation of Neutralizing Antibodies Using a Recombinant C.botulinum Serotype A C Fragment Protein Containing a Six Residue His-Tag

[0694] In Example 27, neutralizing antibodies were generated utilizingthe pHisBotA protein, which contains a histidine-tagged N-terminalextension comprising 10 histidine residues. To determine if thegeneration of neutralizing antibodies is dependent on the presence ofthis particular his-tag, a protein containing a shorter N-terminalextension (comprising 6 histidine residues) was produced and tested forthe ability to generate neutralizing antibodies. This example involveda) the cloning and expression of the p6HisBotA(syn) protein and b) thegeneration and characterization of hyperimmune serum.

[0695] a) Cloning and Expression of the p6HisBotA(syn) Protein

[0696] The p6HisBotA(syn) construct was generated as described below;the term “syn” designates the presence of synthetic gene sequences. Thisconstruct expresses the C frgament of the C. botulinum serotype A toxinwith a histidine-tagged N terminal extension having the followingsequence: MetH is His H is His H is HisMetAla (SEQ ID NO:32); the aminoacids encoded by the botulinal C fragment gene are underlined and thevector encoded amino acids are presented in plain type.

[0697] 6×His oligonucleotides [5′-TATGCATCACCATCACCATCA-3′ (SEQ IDNO:33) and 5′-CATGTGATGGTGATGGTGATGCA-3′ (SEQ ID NO:34) were annealed asfollows. One microgram of each oligonucleotide was mixed in total of 20μl 1× reaction buffer 2 (NEB) and the mixture was heated at 70° C. for 5min and then incubated at 42° C. for 5 min. The annealedoligonucleotides were then ligated with gel purified NdeI/HindIIIcleaved pET23b (T7 promoter) or pET21b (T7lac promoter) DNA and the gelpurified NcoI/HindIII C. botulinum serotype A C fragment synthetic genefragment derived from pAlterBot (Ex. 22). Recombinant clones wereisolated and confirmed by restriction digestion. The DNA sequenceencoding the 6×his-tagged BotA protein contained within p6HisBotA(syn)is listed in SEQ ID NO:35. The amino acid sequence of the p6XHisBotAprotein is listed in SEQ ID NO:36.

[0698] The resulting recombinant p6XHisBotA plasmid was transformed intothe BL21(DE3) pLysS strain, and 1 liter cultures were grown, induced andharvested as described in Example 28. His-tagged protein was purified asdescribed in Example 28, with the following modifications. The bindingbuffer (BB) contained 5 mM imidazole rather than 40 mM imidazole andNP40 was added to the soluble lysate to a final concentration of 0.1%.The bound material was washed on the column with BB until the baselinewas established, then the column was washed successively with BB+20 mMimidazole and BB+40 mM imidazole. The column was eluted as described inExample 28.

[0699] In the case of the pET23-derived expression system, high levelexpression of insoluble 6HisBotA protein was induced. The pET21-derivedvector expressed lower levels of soluble protein that bound the NiNTAresin and eluted in the 40 mM imidazole wash rather than during the lowpH elution. These results (i.e., low level expression of a solubleprotein) are consistent with the results obtained with pHisBotA protein(Ex. 25); the pHisBotA construct, like the pET21-derived vector,contains the T7lac rather than T7 promoter.

[0700] The 6HisBotA protein thus elutes under less stringent conditionsthan the 10× histidine-containing pHisBot protein (100-200 mM imidazole;Ex. 25) presumably due to the reduction in the length of the his-tag.The eluted protein was of the predicted size [i.e., slightly reduced incomparison to pHisBotA protein].

[0701] b) Generation and Characterization of Hyperimmune Serum

[0702] Eight BALBc mice were immunized with purified 6HisBotA proteinusing Gerbu GMDP adjuvant (CC Biotech). The 40 mM imidazole elution wasmixed with Gerbu adjuvant and used to immunize mice. Each mouse receiveda subcutaneous injection of 100 μl antigen/adjuvant mix (12 μg antigen+1μg adjuvant) on day 0. Mice were subcutaneously boosted as above on day14 and bled on day 28. Control mice received pHisBotB protein (preparedas described in Ex. 35 below) in Gerbu adjuvant.

[0703] Anti-C. botulinum serotype A toxoid titers were determined inserum from individual mice from each group using the ELISA described inExample 23a with the exception that the initial testing serum dilutionwas 1:100 in blocking buffer containing 0.5% Tween 20, followed byserial 5-fold dilutions into this buffer. The results of the ELISAdemonstrated that seroconversion (relative to control mice) occurred inall 8 mice.

[0704] The ability of the anti-C. botulinum serotype A C fragmentantibodies present in serum from the immunized mice to neutralize nativeC. botulinum type A toxin was tested using the mouse neutralizationassay described in Example 23b. The amount of neutralizing antibodiespresent in the serum of the immunized mice was determined using serumantibody titrations. The various serum dilutions (0.01 ml) were mixedwith 5 LD₅₀ units of C. botulinum type A toxin and the mixtures wereinjected IP into mice. The neutralizations were performed in duplicate.The mice were then observed for signs of botulism for 4 days. Undilutedserum was found to protect 100% of the injected mice while the 1:10diluted serum did not. This corresponds to a neutralization titer of0.05-0.5 IU/ml.

[0705] These results demonstrate that neutralizing antibodies wereinduced when the 6HisBotA protein was utilized as the immunogen.Furthermore, these results demonstrate that seroconversion and thegeneration of neutralizing antibodies does not depend on the specific Nterminal extension present on the recombinant C. botulinum type A Cfragment proteins.

Example 30 Construction of Vectors for the Expression of His-Tagged C.botulinum Type A Toxin C Fragment Protein Using the Synthetic Gene

[0706] A number of expression vectors were constructed which containedthe synthetic C. botulinum type A toxin C fragment gene. Theseconstructs vary as to the promoter (T7 or T7lac) and repressor elements(lacIq) present on the plasmid. The T7 promoter is a stronger promoterthan is the T7lac promoter. The various constructs provide varyingexpression levels and varying levels of plasmid stability. This exampleinvolved a) the construction of expression vectors containing thesynthetic C. botulinum type A C fragment gene and b) the determinationof the expression level achieved using plasmids containing either thekanamycin resistance or the ampicillin resistance genes in small scalecultures.

[0707] a) Construction of Expression Vectors Containing the Synthetic C.botulinum Type A C Fragment Gene

[0708] Expression vectors containing the synthetic C. botulinum type A Cfragment gene were engineered to utilize the kanamycin resistance ratherthan the ampicillin resistance gene. This was done for several reasonsincluding concerns regarding the presence of residual ampicillin inrecombinant protein derived from plasmids containing the ampicillinresistance gene. In addition, ampicillin resistant plasmids are moredifficult to maintain in culture; the β-lactamase secreted by cellscontaining ampicillin resistant plasmids rapidly degrades extracellularampicillin, allowing the growth of plasmid-negative cells.

[0709] A second altered feature of the expression vectors is theinclusion of lacIq gene in the plasmid. This repressor lowers expressionfrom lac regulated promoters (the chromosomally located, lactoseregulated T7 polymerase gene and the plasmid located T7lac promoter).This down regulates uninduced protein expression and can enhance thestability of recombinant cell lines. The final alteration to the vectorsis the inclusion of either the T7 or T7lac promoters that drive high ormoderate level expression of recombinant protein, respectively.

[0710] The expression plasmids were constructed as follows. In allcases, the protein expressed is the pHisBotA(syn) protein previouslydescribed, and the only differences between constructs is the alterationof the various regulatory elements described above.

[0711] i) Construction of pHisBotA(syn) kan T7lac

[0712] The pHisBotA(syn) kan T7lac construct was made by inserting theSapI/XhoI fragment containing the C. botulinum type A C fragment frompHisBotA(syn) into pET24 digested with SapI/XhoI (Novagen; fragmentcontains kan gene and origin of replication). The desired construct wasselected for kanamycin resistance and confirmed by restrictiondigestion.

[0713] ii) Construction of pHisBotA(syn) kan lacIq T7lac

[0714] The pHisBotA(syn) kan lacIq T7lac construct was made by insertingthe XbaI/HindIII fragment containing the C. botulinum type A C fragmentfrom pHisBotA(syn)kanT7lac into the pET24a vector digested withXbaI/HindIII. The resulting construct was confirmed by restrictiondigestion.

[0715] iii) Construction of pHisBotA(syn) kan lacIq T7

[0716] The pHisBotA(syn) kan lacIq T7 construct was made by insertingthe XbaI/HindIII fragment containing the C. botulinum type A C fragmentfrom pHisBotA(syn) kan lacIq T7lac into XbaI/HindIII-digestedpHisBotB(syn) kan lacIq T7 (described in Ex 37c below). The resultingconstruct was confirmed by restriction digestion.

[0717] b) Determination of the Expression Level Achieved Using PlasmidsContaining Either the Kanamycin Resistance or the Ampicillin ResistanceGenes in Small Scale Cultures

[0718] One liter cultures of pHisBotA(syn) kan T7lac/B1121(DE3)pLysS andpHisBotA(syn) amp T7lac/B121(DE3)pLysS [this is the previouslydesignated pHisBotA(syn) construct] were grown, induced and his-taggedproteins were purified as described in Example 28. No differences inyield or protein integrity/purity were observed.

[0719] These results demonstrate that the antigen induction levels fromexpression constructs were not affected by the choice of ampicillinversus kanamycin antibiotic resistance genes.

Example 31 Fermentation of Cells Expressing Recombinant BotulinalProteins

[0720] a) Fermentation Culture of Cells Expressing Recombinant BotulinalProteins

[0721] Fermentation cultures were grown under the following conditionswhich were optimized for growth of the BL21(DE3) strains containing pETderived expression vectors. An overnight 1 liter feeder culture wasprepared by inoculating of 1 liter media (in a 2 L shaker flask) with afresh colony grown on an LB kan plate. The feeder culture contained: 600mls nitrogen source [20 gm yeast extract (BBL) and 40 gm tryptone(BBL)/600 mls], 200 mls 5× fermentation salts (per liter: 48.5 gmK₂HPO₄, 12 gm NaH₂PO₄.H₂O, 5 gm NH₄Cl, 2.5 gm NaCl), 180 mls dH₂O, 20mls 20% glucose, 2 mls 1 M MgSO₄, 5 mls 0.05M CaCl₂ and 4 mls of a 10mg/ml kanamycin stock. All solutions were sterilized by autoclaving,except the kanamycin stock which was filter sterilized.

[0722] An aliquot (5 ml) of the feeder culture broth was removed priorto inoculation, and grown for 2 days at 37° C. as a culture brothsterility control. Growth was not observed in this control culture inany of the fermentations performed.

[0723] The inoculated feeder culture was grown for 12-15 hrs (ON) at30-37° C. Care was taken to prevent oversaturation of this culture. Thesaturated feeder culture was added to 10 L of fermentation media infermenter (BiofloIV, New Brunswick Scientific, Edison, N.J.) as follows.The fermenter was sterilized 120 min at 121° C. with dH₂O. The sterilewater was removed, and fermentation media added as follows: 6 litersnitrogen source, 2 liters 5× fermentation salts, 2 liters 2% glucose, 20mls 1 M MgSO₄, 50 mls 0.05 M CaCl₂, 2.5-3.5 mls Macol P 400 antifoam(PPG Industries Inc., Gurnee, Ill.), 40 mls 10 mg/ml kanamycin and 10mls trace elements (8 gm FeSO₄.7H₂O, 2 gm MnSO₄.H₂O, 2 gm AlCl₃.6H₂O,0.8 gm CoCl.6H₂O, 0.4 gm ZnSO₄.7H₂O, 0.4 gm Na₂MoO₄.2H₂O, 0.2 gmCuCl₂.2H₂O, 0.2 gm NiCl₂, 0.1 gm H₃BO₄/200 mls 5 M HCl). All solutionswere sterilized by autoclaving, except the kanamycin stock which wasfilter sterilized. Fermentation media was prewarmed to 37° C. before theaddition of the feeder culture.

[0724] After the addition of the feeder culture, the culture wasfermented at 37° C., 400 rpm agitation, and 10 l/mm air sparging. TheDO₂ control was set to 20% PID and dissolved oxygen levels werecontrolled by increasing the rate of agitation from 400-850 rpm underDO₂ control. DO₂ levels were maintained at greater than or equal to 20%throughout the entire fermentation. When agitation levels reached500-600 rpm the temperature was lowered to 30° C. to reduce the oxygenconsumption rate. Culture growth was continued until endogenous carbonsources were depleted. In these fermentations, glucose was depletedfirst [monitored with a glucose monitoring kit (Sigma)], followed byassimilation of acetate and other acidic carbons [monitored using anacetate test kit (Boehringer Mannheim)]. During the assimilation phase,the pH rose from 6.6-6.8 (starting pH) to 7.4-7.5, at which time thebulk of the remaining carbon source was depleted. This was signaled by adrop in agitation rate (from a maximum of 700-800 rpm) and a rise in DO₂levels >30%. This corresponds to a OD₆₀₀ reading of 18-20/ml. At thispoint a fed batch mode was initiated, in which a feed solution of 50%glucose was added at a rate of approximately 4 gm glucose/liter/hr. ThepH was adjusted to 7.0 by the addition of 25% H₃PO₄ (approximately 60mls). Culture growth was continued and reached peak oxygen consumptionwithin the next 3 hrs of growth (while the remaining residualnon-glucose carbon sources were assimilated). This phase ischaracterized by a slow increase in pH, and air sparging was increasedto 15 L/min, to keep the maximum rpm below 850.

[0725] Once the residual acidic carbon sources are depleted theagitation rate decreases to 650-750 rpm and the pH begins to drop. pHcontrol was maintained at 7.0 PID by regulated pump addition of asterile 4M NaOH solution which was consumed at a steady rate for theremainder of the fermentation. Growth was continued at 30° C., and thecultures were grown linearly at a growth rate of 4-7 OD₆₀₀ units/hr, toat least 81.5 OD₆₀₀ units/ml (>30 g/l dry cell weight) withoutinduction. Antifoam (a 1:1 dilution with filter sterilized 100% ethanol)was added as necessary throughout the fermentation to prevent foaming.

[0726] During the fed batch mode, glucose was assimilated immediately(concentration in media consistently less than 0.1 gm/liter) and acetatewas not produced in significant levels by the pET plasmid/BL21(DE3) celllines tested (approximately 1 gm/liter at end of fermentation; this islower than that observed in harvests from shaker flask culturesutilizing the same strains). This was fortuitous, since high levels ofacetate has been shown to inhibit induction levels in a variety ofexpression systems. The above described conditions were found to behighly reproducible between fermentations and utilizing differentexpression plasmids. As a result, glucose and acetate level monitoringwere no longer preformed during fermentation.

[0727] b) Induction of Fermentation Cultures

[0728] Induction with IPTG (250 mg-10 gms, depending on the expressionvector and experiment) was initiated 1-3 hrs after initiation of theglucose feed (30-50 OD₆₀₀/ml). The growth rate after induction wasmonitored on a hourly basis. Aliquots (5-10 ml) of cells were harvestedat the time of induction, and at hourly intervals post-induction.Optical density readings were determined by measuring the absorbance at600 nm of 10 μl culture in 990 μl PBS versus a PBS control. The growthrate after induction was found to vary depending on the expressionsystem utilized.

[0729] c) Monitoring of Fermentation Cultures

[0730] Fermentation cultures were monitored using the following controlassays.

[0731] i) Colony Forming Ability

[0732] An aliquots of cells were removed from the cultures at eachtimepoint sampled (uninduced and at various times after induction) wereserially diluted in PBS (dilution 1=15 μl cells/3 ml PBS, dilution 2=15μl of dilution ⅓ ml PBS, dilution 3=3 or 6 μl of dilution ⅔ mls PBS) and100 μl of dilution 3 was plated on an LB or TSA (trypticase soy agar)plate. The plates were incubated ON at 37° C. and then the colonies arecounted and scored for macro or micro growth.

[0733] ii) Phenotypic Characterization

[0734] Colonies growing on LB or TSA plates (above) from uninduced andinduced timepoints were replica plated onto LB+kan, LB+chloramphenicol(for fermentations utilizing LysS or pACYCGro plasmids), LB+kan+1 mMIPTG and LB plates, in this order. The plates were grown 6-8 hrs at 37°C. and growth was scored on each plate for a minimum of 40-50 wellisolated colonies. The percentage of cells retaining the plasmid at timeof induction (i.e., uninduced cultures immediately prior to the additionof IPTG) was determined to be the # colonies LB+Kan (orchlorarnphenicol) plate/# colonies LB plate X 100%. The percentage ofcells with mutated pET plasmids was determined to be the # coloniesLB+Kan+IPTG plate/# colonies LB plate X 100%. Colonies on all LB plateswere scored morphologically for E. coli phenotype as a contaminationcontrol. Morphologically detectable contaminant colonies were notdetected in any fermentation.

[0735] iii) Recombinant BotA Protein Induction

[0736] A total of 10 OD₆₀₀ units of cells (e.g., 200 μl of cells atOD₆₀₀=50/ml) were removed from each timepoint sample to a 1.5 mlmicrofuge tube and pelleted for 2 min at maximum rpm in a microfuge. Thepellets were resuspended in 1 ml of 50 mM NaHPO₄, 0.5 M NaCl, 40 mMimidazole buffer (pH 6.8) containing 1 mg/ml lysozyme. The samples wereincubated for 20 min at room temperature and stored ON at −70° C.Samples were thawed completely at room temperature and sonicated 2×10seconds with a Branson Sonifier 450 microtip probe at # 3 power setting.The samples were centrifuged for 5 min at maximum rpm in a microfuge.

[0737] An aliquot (20 μl) of the protein samples were removed to 20 μl2× sample buffer, before or after centrifugation, for total and solubleprotein extracts, respectively. The samples were heated to 95° C. for 5min, then cooled and 5 or 10 μl were loaded onto 12.5% SDS-PAGE gels.High molecular weight protein markers (BioRad) were also loaded to allowfor estimation of the MW of identified fusion proteins. Afterelectrophoresis, protein was detected either generally by staining gelswith Coomassie blue, or specifically, by blotting onto nitrocellulose(as described in Ex. 28) for Western blot detection of specifichis-tagged proteins utilizing a NiNTA-alkaline phosphatase conjugateexactly as described by the manufacturer (Qiagen).

[0738] iv) Recombinant Antigen Purification

[0739] At the end of each fermentation run, 1-10 liters of culture wereharvested from the fermenter and the bacterial cells were pelleted bycentrifugation at 6000 rpm for 10 min in a JA10 rotor (Beckman). Thecell pellets were stored frozen at −70° C. or utilized immediatelywithout freezing. Cell pellets were resuspended to 15-20% weight tovolume in resuspension buffer (generally 50 mM NaPO₄, 0.5 M NaCl, 40 mMimidazole, pH 6.8) and lysed utilizing either sonication or highpressure homogenization.

[0740] For sonication, the resuspension buffer was supplemented withlysozyme to 1 mg/ml, and the suspension was incubated for 20 min. atroom temp. The sample was then frozen ON at −70° C., thawed andsonicated 4×20 seconds at microtip maximum to reduce viscosity. Forhomogenization, the cells were lyzed by 2 passes through a homogenizer(Rannie Mini-lab type 8.30H) at 600 Bar. Cell lysates were clarified bycentrifugation for 30 min at 10,000 rpm in a JA10 rotor.

[0741] For IDA chromatography, samples were flocculated utilizingpolyethyleneimine (PEI) prior to centrifugation. Cell pellets wereresuspended in cell resuspension buffer (CRB: 50 mM NaPO₄, 0.5 M NaCl,40 mM imidazole, pH 6.8) to create a 20% cell suspension (wet weight ofcells/volume of CRB) and cell lysates were prepared as described above(sonication or homogenization). PEI (a 2% solution in dH₂O, pH 7.5 withHCl) was added to the cell lysate a final concentration of 0.2%, andstirred for 20 min at room temperature prior to centrifugation (8,500rpm in JA10 rotor for 30 minutes at 4° C.). This treatment removed RNA,DNA and cell wall components, resulting in a clarified, low viscositylysate (“PEI clarified lysate”).

[0742] His-tagged proteins were purified from soluble lysates bymetal-chelate affinity chromatography using either a NiNTA resin (asdescribed in Ex. 28) or an IDA (iminodiacetic acid) resin as describedbelow.

[0743] IDA resin affinity purifications were performed utilizing a lowpressure chromatography system (ISCO). A 7 ml (small scale) or 70 ml(large scale) Chelating Sepharose Fast Flow (Pharmacia) affinity columnwas poured; in addition, a second guard column was poured and attachedin line with the first column (to capture Ni ions that leached off theaffinity column). The columns were washed with 3 column volumes of dH₂O.The guard column was then removed and the affinity column was washedwith 0.3 M NiSO₄ until resistivity was established, then with dH₂O untilthe resistivity returned to baseline. The columns were reconnected andequilibrated with cell resuspension buffer (CRB; 50 mM NaPO₄, 0.5 MNaCl, 40 mM imidazole, pH 6.8). The clarified sample (in CRB) wasloaded. Flow rates were 5 ml/min for small scale columns and 20 m/minfor large scale columns. After sample loading, the column was washedwith CRB until a baseline established and bound protein was eluted withelution buffer (50 mM NaPO₄, 0.5 M NaCl, 800 mM imidazole, 20% glycerol,pH 6.8 or 8.0). Protein samples were stored at 4° C. or −20° C. Theyield of eluted protein was established by measuring the OD₂₈₀ of theelutions, with a 1 mg/ml solution of protein assumed to yield anabsorbance reading of 2.0.

[0744] The IDA columns may be regenerated and reused multiple times(>10). To regenerate the column, the column was washed with 2-3 columnvolumes of H₂O, then 0.05 M EDTA until all of the blue/green color wasremoved followed by a wash with dH₂O. The IDA columns were sterilizedwith 0.1 M NaOH (using at least 3 column volumes but not more than 50minutes contact time with column packing material), then washed with 3column volumes 0.05 M NaPO₄, pH 5.0, then dH₂O and stored at roomtemperature in 20% ethanol.

Example 32 Construction of a Folding Chaperone Overexpression System

[0745] Co-overexpression of the E. coli GroEL/GroES folding chaperonesin a cell expressing a recombinant foreign protein has been reported toenhance the solubility of some foreign proteins that are otherwiseinsoluble when expressed in E. coli [Gragerouu et al. (1992) Proc. Natl.Acad. Sci. USA 89:10344]. The improvement in solubility is thought to bedue to chaperone-mediated binding and unfolding of insoluble denaturedproteins, thus allowing multiple attempts for productive refolding ofrecombinant proteins. By overexpressing the chaperones, theunfolding/refolding reaction is driven by excess chaperone, resulting,in some cases, in higher yields of soluble protein.

[0746] In this example, a chaperone overexpression system, compatiblewith pET vector expression systems, was constructed to facilitatetesting chaperone-mediated solubilization of C. botulinum type Aproteins. This example involved the cloning of the GroEL/ES operon andconstruction of a pLysS-based chaperone hyperexpression system.

[0747] The GroEL/GroES operon was PCR amplified and cloned into thepCRScript vector as described in Example 28. The following primer pairwas used: 5′-CGCAT ATGAATATTCGTCCATTGCATG-3′ (SEQ ID NO:37) [5′ primer,start codon of groES gene converted to NdeI site (underlined)] and5′-GGAAGCTTGCAGGGCAAT TACATCATG (SEQ ID NO:38) (3′ primer, stop codon ofgroEL gene italicized, engineered HindIII site underlined). Followingamplification, the chaperone operon was excised as an NdeI/HindIIIfragment and cloned into pET23b digested with NdeI and HindIII. Thisconstruction places the Gro operon under the control of the T7 promoterof the pET23 vector. The desired construct was confirmed by restrictiondigestion.

[0748] The T7 promoter-Gro operon-T7 terminator expression cassette wasthen excised as a BglII/BspEI (filled) fragment and cloned into BamHI(compatible with BglII)/HindIII (filled) cleaved pLysS plasmid (thisremoved the T7 lysozyme gene). The resulting construct was designatedpACYCGro, since the plasmid utilizing the pACYC184 origin from the plysSplasmid. Proper construction was confirmed by restriction digestion.

[0749] pACYCGro was transformed into BL21(DE3), cultures were grown andinduced with 1 mM IPTG as described in preceding examples. Total andsoluble protein extracts were generated from cells removed before andafter IPTG induction and were resolved on a 12.5% SDS-PAGE gel andstained with Coomassie blue. This analysis revealed that high levels ofsoluble GroE1 and GroES proteins were made in the induced cells. Theseresults demonstrated that the chaperone hyper-expression system wasfunctional.

Example 33 Growth of BotA/pACYCGro Cell Lines in Fermentation Cultures

[0750] Induction of BL21(DE3) cells lacking the LysS plasmid whichcontained BotA expression constructs grown in shaker flask orfermentation culture resulted in the expression of primarily insolubleBotA protein. Fermentation cultures were performed to determine if thesimultaneous overexpression of the Gro operon and recombinant C.botulinum type A proteins (BotA proteins) resulted in enhancedsolubility of the recombinant BotA protein. This example involved thefermentation of pHisBotA(syn)kan lacIq T7lac/pACYCGro BL21(DE3) andpHisBotA(syn)kan lacIq T7/pACYCGro BL21(DE3) cell lines. Thefermentations were repeated exactly as described in Example 31.Chloramphenicol (34 μg/ml) was included in the feeder and fermentationcultures.

[0751] a) Fermentation of pHisBotA(syn)kan lacIq T7lac/pACYCGroBL21(DE3) Cells

[0752] For fermentation of cells containing plasmids comprising theT7lac promoter, induction was with 2 gms IPTG at 1 hr post initiation ofglucose feed. The OD₆₀₀ was 35 at time of induction, then 48.5, 61.5, 67at 1-3 hrs post induction. Viable colony counts decreased from 0-3 hrinduction [21 (13), 0, 0, 0; dilution 3 utilized 3 μl of dilution 2cells] with numbers in parenthesis for the indicating microcolonies. Of28 colonies scored at the time of induction, 23 retained thepHisBotA(syn)kan lacIq T7lac plasmid (kan resistant), 22 contained thechaperone plasmid (chloramphenicol resistant) and no colonies atinduction grew on IPTG+Kan plates (no mutations detected). These resultswere indicative of very strong promoter induction, since colonyviability dropped immediately after induction.

[0753] Total and soluble extracts were resolved on a 12.5% SDS-PAGE geland stained with Coomassie. High level induction of Gro chaperones wasobserved, but very low level expression of soluble BotA protein wasobserved, increasing from 1 to 4.0 hrs post induction (no expressiondetected in uninduced cells). The dramatically lower expression of theBotA antigen in the presence of chaperone may be due to promoterocclusion (i.e., the stronger T7 promoter on the chaperone plasmid ispreferentially utilized).

[0754] b) Fermentation of pHisBotA(syn)kan lacIq T7/pACYCGro BL21(DE3)Cells

[0755] A fermentation utilizing the T7-driven BotA expression plasmidwas performed. Induction was with 1 gm IPTG at 2 hrs post initiation ofglucose feed. The OD₆₀₀ was 41 at time of induction, then 51.5, 61.5,61.5 and 66 at 1-4 hrs post induction. Viable colony counts decreasedfrom 0-4 hrs induction [71, 1 (34), 1 (1), 1, 0; dilution 3 utilized 6μl dilution 2 cells) with numbers in parenthesis for the uninducedtimepoint indicating microcolonies. Of 65 colonies scored at the time ofinduction, all 65 retained both the pHisBotA(syn)kan lacIq T7 plasmid(kan resistant) and the chaperone plasmid (chloramphenicol resistant)and no colonies at induction grew on IPTG+Kan plates (no mutationsdetected).

[0756] Total and soluble extracts were resolved on a 12.5% SDS-PAGE geland stained with Coomassie. High level induction of Gro chaperones andmoderate level expression of soluble BotA protein was observed,increasing from 1 to 4.0 hrs post induction (no expression detected inuninduced cells).

[0757] A PEI-clarified lysate (0.2% final cocnentration PEI) [850 mlfrom 130 gm cell pellet (2 liters fermentation harvest)] was purified ona large scale IDA column. A total of 78 mg of protein was eluted.Extracts from the purification were resolved on a 12.5% SDS-PAGE gel andstained with Coomassie. The elution was found to contain anapproximately 1:1 mix of BotA/chaperone protein (FIG. 32). PEI lysatesprepared in this manner were typically 16 OD₂₈₀/ml. This was estimatedto be 8 mg protein/ml of lysate (by BCA assay). Thus, the elutedrecombinant BotA protein represented 0.55% of the total soluble cellularprotein applied to the column.

[0758] In FIG. 32, lane 1 contains molecular weight markers, lanes 2-9contain extracts from pHisBotA(syn)kan lacIq T7/pACYCGro/BL21(DE3) cellsbefore or during purification on the IDA column. Lane 2 contains totalprotein extract; lane 3 contains soluble protein extract; lanes 4 and 5contain PEI-clarified lysates (duplicates); lanes 6 and 7 containflow-through from the IDA column (duplicates) and lanes 8 and 9 containIDA column elute (lane 9 contains 1/10 the amount applied to lane 8).

[0759] These results demonstrate, that although the majority of the BotAprotein produced was insoluble, 20 mg/liter of soluble recombinant BotAprotein can be purified utilizing the pHisBotA(syn)kan lacIq17/pACYCGto/BL21(DE3) expression system.

Example 34 Purification of Recombinant BotA Protein From FoldingChaperones

[0760] In this example of size exclusion chromatography was used topurify the recombinant BotA protein away from the folding chaperones andimidazole present in the IDA-purified material (Ex. 33).

[0761] To enhance the solubility of the recombinant BotA protein duringscale-up, the protein was co-expressed with folding chaperones (Ex. 33).As observed with the recombinant BotB protein (Example 40 below), thefolding chaperones co-eluted with the recombinant BotA protein duringthe Ni-IDA purification step. Because the recombinant BotA and BotBproteins have similar molecular weights (about 1/10 the size of thenon-reduced folding chaperone) and the imidazole step gradient strategywas unsuccessful in purifying BotB away from the folding chaperone (seeEx. 40), size exclusion chromatography was examined for the ability topurify the recombinant BotA protein away from the folding chaperones.

[0762] A column (2.5×24 cm) containing Sephacryl S-100 HR (Pharmacia)was poured (bed volume˜110 ml). Proteins having molecular weightsgreater than 100 K are expected to elute in the void volume under theseconditions and smaller proteins should be retained by the beads andelute at different times, depending on their molecular weights. Tomaintain solubility of the purified BotA protein, the Sephacryl columnwas equilibrated in a buffer having the same salt concentration as thebuffer used to elute the BotA protein from the IDA column (i.e., 50 mMsodium phosphate, 0.5 M NaCl, 10% glycerol; all reagents fromMallinkrodt, Chesterfield, Mo.).

[0763] Five milliliters of the IDA-purified recombinant BotA protein(Ex. 33) was filtered through a 0.45μ syringe filter, applied to thecolumn and the equilibration buffer was pumped through the column at aflow rate of 1 ml/minute. Eluted proteins were monitored by absorbanceat 280 nm and collected either manually or with a fraction collector(BioRad). Appropriate fractions were pooled, if necessary, and theprotein was quantitated by absorbance at 280 run and/or BCA proteinassay (Pierce). The isolated peaks were then analyzed by native and/orSDS-PAGE to identify the proteins present and to evaluate purity. Thefolding chaperone eluted first, followed by the recombinant BotA proteinand then the imidazole peak.

[0764] SDS-PAGE analysis (12.5% polyacrylamide, reduced samples) wasused to evaluate the purity of the IDA-purified recombinant BotA proteinbefore and after S-100 purification. FIG. 33 shows the difference inpurity before and after the S-100 purification step. In FIG. 33, lane 1contains molecular weight markers (BioRad broad range). Lane 2 shows theIDA-purified recombinant BotA protein preparation, which is contaminatedwith significant amounts of the folding chaperone. Following S-100purification, the amount of folding chaperone present in the BotA sampleis reduced dramatically (lane 3). Lane 4 contains no protein (i.e., itis a blank lane); lanes 5-8 contain samples of IDA-purified recombinantBotB and BotE proteins and are discussed infra.

[0765] Endotoxin levels in the S-100 purified BotA preparation weredetermined using the LAL assay (Associates of Cape Cod) as describe inExample 24. The purified BotA preparation was found to contain 22.7 to45.5 EU/mg recombinant protein.

[0766] These results demonstrate that size exclusion chromatography wassuccessful in purifying the recombinant BotA protein from foldingchaperones and imidazole following an initial IDA purification step.Furthermore, these results demonstrate that the S-100 purified BotAprotein was substantially free of endotoxin.

Example 35 Cloning and Expression of the C Fragment of the C. botulinumSerotype B Toxin Gene

[0767] The C. botulinum type B neurotoxin gene has been cloned andsequenced [Whelan et al. (1992) Appl. Environ. Microbiol. 58:2345 andHutson et al. (1994) Curr. Microbiol. 28:101]. The nucleotide sequenceof the toxin gene derived from the Eklund 17B strain (ATCC 25765) isavailable from the EMBL/GenBank sequence data banks under the accessionnumber X71343; the nucleotide sequence of the coding region is listed inSEQ ID NO:39. The amino acid sequence of the C. botulinum type Bneurotoxin derived from the strain Eklund 17B is listed in SEQ ID NO:40.The nucleotide sequence of the C. botulinum serotype B toxin genederived from the Danish strain is listed in SEQ ID NO:41 and thecorresponding amino acid sequence is listed in SEQ ID NO:42.

[0768] The DNA sequence encoding the native C. botulinum serotype B Cfragment gene derived from the Eklund 17B strain can be expressed usingthe pETHisb vector; the resulting coding region is listed in SEQ IDNO:43 and the corresponding amino acid sequence is listed in SEQ IDNO:44. The DNA sequence encoding the native C. botulinum serotype B Cfragment gene derived from the Danish strain can be expressed using thepETHisb vector; the resulting coding region is listed in SEQ ID NO:45and the corresponding amino acid sequence is listed in SEQ ID NO:46. TheC frgament region from any strain of C. botulinum serotype B can beamplified and expressed using the approach illustrated below using the Cfragment derived from C. botulinum type B 2017 strain.

[0769] The C. botulinum type B neurotoxin gene is synthesized as asingle polypeptide chain which is processed to form a dimer composed ofa light and a heavy chain linked via disulfide bonds; the type Bneurotoxin has been reported to exist as a mixture of predominatlysingle chain with some double chain (Whelan et al., supra). The 50 kDcarboxy-terminal portion of the heavy chain is referred to as the Cfragment or the H_(C) domain. Expression of the C fragment of C.botulinum type B toxin in heterologous hosts (e.g., E. coli) has notbeen previously reported.

[0770] The native C fragment of the C. botulinum serotype B toxin genewas cloned and expression constructs were made to facilitate proteinexpression in E. coli. This example involved PCR amplification of thegene, cloning, and construction of expression vectors.

[0771] The C fragment of the C. botulinum serotype B (BotB) toxin genewas cloned using the protocols and conditions described in Example 28for the isolation of the native BotA gene. The C. botulinum type B 2017strain was obtained from the American Type Culture Collection (ATCC#17843). The following primer pair was used to amplify the BotB gene:5′-CGCCATGGCTGATACAATACTAATAGAA ATG-3′ [5′ primer, engineered NcoI siteunderlined (SEQ ID NO:47)] and 5′-GCAAG CTTTTATTCAGTCCACCCTTCATC-3′ [3′primer, engineered HindIII site underlined, native gene terminationcodon italicized (SEQ ID NO:48)]. After cloning into the pCRscriptvector, the NheI(filled)/HindIII fragment was cloned into pETHisb vectoras described for BotA C fragment gene in Example 28. The resultingconstruct was termed pHisBotB.

[0772] pHisBotB expresses the BotB gene sequences under thetranscriptional control of the T7 lac promoter and the resulting proteincontains an N-terminal 10×His-tag affinity tag. The pHisBotB expressionconstruct was transformed into BL21 (DE3) pLysS competent cells and 1liter cultures were grown, induced and his-tagged proteins were purifiedutilizing a NiNTA resin (eluted in low pH elution buffer) as describedin Example 28. Total, soluble and purified proteins were resolved bySDS-PAGE and detected by Coomassie staining and Western blothybridization utilizing a chicken anti-C. botulinum serotype B toxoidprimary antibody (generated by immunization of hens using C. botulinumserotype B toxoid as described in Example 3). Samples of BotA and BotE Cfragment proteins were included on the gels for MW and immunogenicitycomparisons. Strong immunoreactivity to only the BotB protein wasdetected with the anti-C. botulinum serotype B toxoid antibodies. Therecombinant BotB protein was expressed at low levels (3 mg/liter) as asoluble protein. The purified BotB protein migrated as a single band ofthe predicted MW (i.e., ˜50 kD).

[0773] These results demonstrate the cloning of the native C. botulinumserotype B C fragment gene, the expression and purification of therecombinant BotB protein as a soluble his-tagged protein in E. coli.

Example 36 Generation of Neutralizing Antibodies Using the RecombinantpHisBotB Protein

[0774] The ability of the purified pHisBot protein to generateneutralizing antibodies was examined. Nine BALBc mice were immunizedwith BotB protein (purified as described in Ex. 35) using Gerbu GMDPadjuvant (CC Biotech). The low pH elution was mixed with Gerbu adjuvantand used to immunize mice. Each mouse received a subcutaneous injectionof 100 μl antigen/adjuvant mix (15 μg antigen+1 μg adjuvant) on day 0.Mice were subcutaneously boosted as above on day 14 and bled on day 28.Mice were subsequently boosted 1-2 weeks after bleeding and were thenbled on day 70.

[0775] Anti-C. botulinum serotype B toxoid titers were determined in day28 serum from individual mice from each group using the ELISA protocoloutlined in Example 29 with the exception that the plates were coatedwith C. botulinum serotype B toxoid, and the primary antibody was achicken anti-C. botulinum serotype B toxoid. Seroconversion [relative tocontrol mice immunized with pHisBotE antigen (described below)] wasobserved with all 9 mice immunized with the purified pHisBotB protein.

[0776] The ability of the anti-BotB antibodies to neutralize native C.botulinum type B toxin was tested in a mouse-C. botulinum neutralizationmodel using pooled mouse serum (see Ex. 23b). The LD₅₀ of purified C.botulinum type B toxin complex (Dr. Eric Johnson, University ofWisconsin, Madison) was determined by a intraperitoneal (IP) method[Schantz and Kautler (1978), supra] using 18-22 g female ICkr mice. Theamount of neutralizing antibodies present in the serum of the immunizedmice was determined using serum antibody titrations. The various serumdilutions (0.01 ml) were mixed with 5 LD₅₀ units of C. botulinum type Btoxin and the mixtures were injected IP into mice. The neutralizationswere performed in duplicate. The mice were then observed for signs ofbotulism for 4 days. Undiluted serum (day 28 or day 70) was found toprotect 100% of the injected mice while the 1:10 diluted serum did not.This corresponds to a neutralization titer of 0.05-0.5 IU/ml.

[0777] These results demonstrate that seroconversion occurred andneutralizing antibodies were induced when the pHisBotB protein wasutilized as the immunogen.

Example 37 Construction of Vectors to Facilitate Expression ofHis-Tagged BotB Protein in Fermentation Cultures

[0778] A number of expression vectors were constructed to facilitate theexpression of recombinant BotB protein in large scale fermentationculture. These constructs varied as to the strength of the promoterutilized (T7 or T7lac) and the presence of repressor elements (lacIq) onthe plasmid. The resulting constructs varied in the level of expressionachieved and in plasmid stability which facilitated the selection of aoptimal expression system for fermentation scaleup.

[0779] The BotB expression vectors created for fermentation culture wereengineered to utilize the kanamycin rather than the ampicillinresistance gene, and contained either the T7 or T7lac promoter, with orwithout the lacIq gene for the reasons outlined in Example 30.

[0780] In all cases, the protein expressed by the various expressionvectors is the pHisBot B protein described in Example 35, with the onlydifferences between clones being the alteration of various regulatoryelements. Using the designations outlined below, the pHisBotB clone (Ex.35) is equivalent to pHisBotB amp T7lac.

[0781] a) Construction of pHisBotB kan T7lac

[0782] pHisBotB kan T7lac was constructed by insertion of theBglII/HindIII fragment of pHisBotB which contains the BotB genesequences into the pPA1870-2680 kan T7lac vector which had been digestedwith BglII and HindIII (the pPA1870-2680 kan T7lac vector contains thepET24 kan gene in the pET23 vector, such that no lacIq gene is present).Proper construction of pHisBotB kan T7lac was confirmed by restrictiondigestion.

[0783] b) Construction of pHisBotB kan lacIq T7lac

[0784] pHisBotB kan lacIq T7lac was constructed by insertion of theBglII/HindIII fragment of pHisBotB which contains the BotB genesequences into similarly cut pET24a vector. Proper construction ofpHisBotB kan lacIq T7lac was confirmed by restriction digestion.

[0785] c) Construction of pHisBotB kan lacIq T7

[0786] pHisBotB kan lacIq T7 was constructed by inserting the NdeI/XhoIfragment from pHisBotE kan lacIq T7lac which contains the BotB genesequences into similarly cleaved pPA1870-2680 kan lacIq T7 vector (thisvector contains the T7 promoter, the same N-terminal his-tag as the Botconstructs, the C. difficile toxin A insert, and the kan lacIq genes;this cloning replaces the C. dificile toxin A insert with the BotBinsert). Proper construction was confirmed by restriction digestion.

[0787] Expression of recombinant BotB protein from these expressionvectors and purification of the BotB protein is described in Example 38below.

Example 38 Fermentation and Purification of Recombinant BotB ProteinUtilizing the pHisBotB kan lacIq T7lac, pHisBotB kan T7lac and pHisBotBkan lacIq T7 Vectors

[0788] The pHisBotB kan lacIq T7lac, pHisBotB kan T7lac and BotB kanlacIq T7 constructs [all transformed into the B121(DE3) strain] weregrown in fermentation cultures to determine the utility of the variousconstructs for large scale expression and purification of soluble BotBprotein. All fermentations were performed as described in Example 31.

[0789] a) Fermentation of pHisBotB kan lacIq T7lac/B121(DE3) Cells

[0790] The fermentation culture was induced 45 min post start of glucosefeed with 1 gm IPTG (final concentration=0.4 mM). pH was maintained at6.5 rather than 7.0. The OD₆₀₀ was 27 at time of induction, then 35, 38,and 40 at 1-3 hrs post induction. Duplicate platings of diluted 1 hrinduction samples (dilutions were prepared as described Ex. 31, dilution3 utilized 3 μl of dilution 2 cells) on TSA and LB+kan plates yielded 89TSA colonies and 81 kan colonies (90% kan resistant).

[0791] Total and soluble protein extracts were resolved on a 12.5%SDS-PAGE gel and total protein was detected by staining with Coomassieblue. Low level induction of insoluble Bot B protein was observed,increasing from 1 to 3 hrs post induction (no expression was detected inuninduced cells).

[0792] b) Fermentation of pHisBotB kan T7lac/B121(DE3) Cells

[0793] The fermentation culture was induced 1 hr post start of glucosefeed with 2 gm IPTG (final concentration=0.8 mM). pH was maintained at6.5 rather than 7.0. The OD₆₀₀ was 24.5 at time of induction, then 31.5,32, and 33 at 1-3 hrs post induction, respectively. Duplicate platingsof diluted 0 hr and 2 hr induction samples (dilutions were prepared asdescribed Ex. 31; dilution 3 utilized 3 μl of dilution 2 cells) on TSAand LB+kan plates yielded 32 TSA colonies and 54 kan colonies (all kanresistant) for uninduced cells, and 1 TSA colony and 0 kan colonies 2 hrpost induction. These results were indicative of strong induction, sinceviable counts decreased dramatically 2 hrs post induction.

[0794] Total and soluble extracts were resolved on a 10% SDS-PAGE geland total protein was detected by staining with Coomassie blue. Moderateinduction of insoluble BotB protein was observed, increasing from 1 to 3hrs post induction (no expression was detected in uninduced cells).

[0795] c) Fermentation of pHisBotB kan lacIq T7/B121(DE3) Cells

[0796] The fermentation was induced 2 hr post start of glucose feed with4 gm IPTG (final concentration=1.6 mM). pH was maintained at 6.5 ratherthan 7.0. The OD₆₀₀ was 45 at time of induction, then 47, 50, and 50 and55 at 1-4 hrs post induction, respectively. Viable colony countsdecreased after induction (96, 1, 1, 2, 3; dilution 3 utilized 3 μl ofdilution 2 cells). Of 63 colonies scored at the time of induction, all63 retaining the BotB plasmid (kan resistant) and no colonies atinduction grew on IPTG+Kan plates (no mutations detected).

[0797] Total and soluble extracts were resolved on a 12.5% SDS-PAGE geland total protein was detected by staining with Coomassie blue. Moderatelevel induction of insoluble BotB protein was observed, increasing from1 to 4 hrs post induction (lower level expression was detected inuninduced cells, since the T7 rather than T7lac promoter was utilized).

[0798] d) Purification of pHisBotB Protein From pHisBotB ampT7lac/B121(DE3) Cells

[0799] Soluble recombinant BotB protein was purified utilizing NiNTAresin from 80 ml of cell lysate generated from cells harvested from apHisBotB fermentation [using the pHisBotB amp T7lac/B121(DE3) strain].As predicted from the small scale results above, the majority of theinduced protein was insoluble. As well, the eluted material wascontaminated with multiple E. coli contaminant proteins. A Coomassieblue-stained SDS-PAGE gel containing extracts derived from pHisBotB ampT7lac/B121(DE3) cells before and during purification is shown in FIG.34. In FIG. 34, lane 1 contains broad range protein MW markers (BioRad).Lanes 2-5 contain extracts prepared from pHisBotB amp T7lac/B121(DE3)cells grown in fermentation culture; lane 2 contains total protein; lane3 contains soluble protein; lane 4 contains protein which did not bindto the NiNTA column (i.e., the flow-through) and lane 5 contains proteineluted from the NiNTA column.

[0800] Similar results were obtained using a small scale IDA columnutilizing a cell lysate from the pHisBotB kan lacIq T7 fermentationdescribed above. 250 mls of a 20% w/v PEI clarified lysate (50 gms cellpellet) of botB kan lacIq T7/B121(DE3) cells were purified on a smallscale IDA column. The total yield of eluted protein was 21 mg protein(assuming 1 mg/ml solution=2 OD₂₈₀/ml). When analyzed by SDS-PAGE andCoomassie staining, the BotB protein was found to comprise approximately50% of the eluted protein with the remainder being a ladder of E. coliproteins similar to that observed with the NiNTA purification.

[0801] The NiNTA alkaline phosphatase conjugate was utilized to detecthis-tagged proteins on a Western blot containing total, soluble, soluble(PEI clarified), soluble (after IDA column) and elution samples from theIDA column purification. The results demonstrated that a smallpercentage of BotB protein was soluble, that the soluble protein was notprecipitated by PEI treatment and was quantitatively bound by the IDAcolumn. Since a 1 liter fermentation harvest yielded a 67.5 gm cellpellet, this indicated that the yield of soluble affinity purified BotBprotein from the IDA column was 14 mg/liter.

Example 39 Co-Expression of Recombinant BotB Proteins and FoldingChaperones in Fermentation Cultures

[0802] Fermentations were performed to determine if the simultaneousoverexpression of folding chaperones (i.e., the Gro operon) and the BotBprotein resulted in enhanced solubility of the Bot B protein. Thisexample involved fermentation of the pHisBotBkan lacIq T7lac/pACYCGroBL21(DE3), pHisBotB kan T7lac/pACYCGro B121(DE3) and pHisBotBkan lac/qT7/pACYCGro BL21(DE3) cell lines. Fermentation was carried out asdescribed in Example 31; 34 μg/ml chloramphenicol was included in thefeeder and fermentation cultures.

[0803] a) Fermentation of pHisBotBkan lacIq T7lac/pACYCGro BL21(DE3)Cells

[0804] Induction was with 4 gms IPTG at 1 hr 15 min post initiation ofthe glucose feed. The OD₆₀₀ was 38 at time of induction, then 50, 58.5,62 and 68 at 1-4 hrs post induction. Viable colony counts decreasedduring induction (24, 0, 0, 2, 0 at 0-4 hr induction; dilution 3utilized 3 μl of dilution 2 cells). Of 24 colonies scored at the time ofinduction, 24 retained the BotB plasmid (kan resistant), 24 containedthe chaperone plasmid (chloramphenicol resistant) and no colonies atinduction grew on IPTG+Kan plates (no mutations detected).

[0805] Total and soluble extracts were resolved on 12.5% SDS-PAGE gelsand were either stained with Coomassie blue or subjected to Westernblotting (his-tagged proteins were detected utilizing the NiNTA-alkalinephosphatase conjugate). This analysis revealed that the Gro chaperoneswere induced to high levels, but very low level expression of solubleBotB protein was observed, increasing from 1 to 4.0 hrs post induction(no expression detected in uninduced cells, induced protein detectedonly on Western blot). The dramatically lower expression of BotB proteinin the presence of chaperone may be due to promoter occlusion (i.e., thestronger T7 promoter on the chaperone plasmid was preferentiallyutilized).

[0806] b) Fermentation of pHisBotB kan T7lac/pACYCGro/B121(DE3) Cells

[0807] Induction was with 4 gms IPTG at 1 hr post initiation of theglucose feed. The OD₆₀₀ was 33.5 at time of induction, then 44, 51, 58.5and 69 at 1-4 hrs post induction. Viable colony counts decreased after 2hrs induction (43, 65, 74, 0 (70), 0 (70) at 0-4 hr induction; bracketednumbers represent microcolonies; dilution 3 utilized 3 μl of dilution 2cells). Most colonies at induction retained the BotB plasmid (kanresistant) and the chaperone plasmid (chloramphenicol resistant) and nocolonies at induction grew on IPTG+Kan plates (no mutations detected).

[0808] Total and soluble extracts were resolved on a 12.5% SDS-PAGE geland subjected to Western blotting; his-tagged proteins were detectedutilizing the NiNTA-alkaline phosphatase conjugate. This analysisrevealed that the Gro chaperones were induced to high levels and lowlevel expression of soluble Bot B protein was observed, increasing from1 to 4.0 hrs post induction (no expression detected in uninduced cells).

[0809] A small scale IDA purification of BotB protein from a 250 ml PEIclarified 15% w/v extract (37.5 gm cell pellet) yielded approximately12.5 mg protein, of which approximately 50% was BotB protein and 50% wasGroEL chaperone (assessed by Coomassie staining of a 10% SDS-PAGE gel).The NiNTA alkaline phosphatase conjugate was utilized to detecthis-tagged proteins on a Western blot containing total, soluble, soluble(PEI clarified), soluble (after IDA column) and elution samples from theIDA column purification. The results demonstrated that all of the BotBprotein produced by the pHisBotB kan T7lac/pACYCGro/B121(DE3) cells wassoluble; the BotB protein was not precipitated by PEI treatment and wasquantitatively bound by the IDA column. Since a 1 liter fermentationharvest yielded a 75 gm cell pellet, this indicated that the yield ofsoluble affinity purified bot B protein from this fermentation was 12.5mg/liter. These results also demonstrated that additional purificationsteps are necessary to separate the chaperone proteins from the BotBprotein.

[0810] c) Fermentation of pHisBotBkan lacIq T7/pACYCGro/BL21(DE3) Cells

[0811] Induction was with 4 gms IPTG at 2 hr post initiation of theglucose feed. The OD₆₀₀ was 46 at time of induction, then 56, 63, 69 and71.5 at 1-4 hrs post induction. Viable colony counts decreased afterinduction (58, 3(5), 3, 0, 0 at 0-4 hr induction; bracketed numbersrepresent microcolonies; dilution 3 utilized 3 μl of dilution 2 cells).All (53/53) colonies scored at the time of induction retained the BotBplasmid (kan resistant) and the chaperone plasmid (chloramphenicolresistant) and no colonies at induction grew on IPTG+Kan plates (nomutations detected).

[0812] Total and soluble extracts were resolved on a 10% SDS-PAGE gelsand Western blotted and his-tagged proteins were detected utilizing theNiNTA-alkaline phosphatase conjugate. This analysis revealed that theGro chaperones were induced to high levels (observed by ponceau Sstaining), and a much higher expression of soluble Bot B protein(compared to expression in the pHisBotB kan T7lac/pACYCGro fermentation)was observed at all timepoints, including uninduced cells (some increasein BotB protein levels were observed after induction).

[0813] A small scale IDA purification of BotB protein from a 100 ml PEIclarified 15% w/v extract (15 gm cell pellet) yielded approximately 40mg protein, of which approximately 50% was BotB protein and 50% wasGroEL chaperone, as assessed by Coomassie staining of a 10% SDS-PAGEgel. The NiNTA alkaline phosphatase conjugate was utilized to detecthis-tagged proteins on a Western blot containing total, soluble, soluble(PEI clarified), soluble (after IDA column) and elution samples from theIDA column purification. The results demonstrated that a significantpercentage (i.e., ˜10-20%) of BotB protein was soluble, that thesolubilized protein was not precipitated by PEI treatment and wasquantitatively bound by the IDA column. Since a 10 liter fermentationyielded a 108 gm cell pellet, this indicated that the yield of solubleaffinity purified BotB protein from this fermentation was 144 mg/liter.

[0814] In a scale up experiment, 2 liters of a 20% w/v PEI clarifiedlysate of pHisBotB kan lacIq T7/pACYCGro/BL21(DE3) cells were purifiedon a large scale IDA column. The purification was performed induplicate. The total yield of BotB protein was 220 and 325 mgs proteinin the two experiments (assuming 1 mg/ml solution=2.0 OD₂so/ml). Thisrepresents 0.7% or 1.0%, respectively, of the total soluble cellularprotein (assuming a PEI lystate having a concentration of 8 mgprotein/ml and that the eluted material comprises a 1:1 mixture of BotBand folding chaperone). The NiNTA alkaline phosphatase conjugate wasutilized to detect his-tagged proteins on a Western blot containingtotal, soluble, soluble (PEI clarified), soluble (after IDA column) andelution samples from the IDA column purification. These resultsdemonstrated that a significant percentage (i.e., ˜10-20%) of the BotBprotein was soluble, that the solubilized protein was not precipitatedby PEI treatment and was quantitatively bound by the IDA column. Since a1 liter fermentation harvest yielded a 108 gm cell pellet, thisindicated that the yield of soluble affinity purified BotB protein fromthe large scale purification was 60 mg or 89 mg/liter. These resultsalso demonstrated that further purification would be necessary to removethe contaminating chaperone protein.

[0815] The above results provide methodologies for the purification ofsoluble BotB protein from fermentation cultures, in a form contaminatedpredominantly with a single E. coli protein (the folding chaperoneutilized to enhance solubility). In the next example, methods areprovided for the removal of the contaminating chaperone protein.

Example 40 Removal of Contaminating Folding Chaperone Protein FromPurified Recombinant C. botulinum Type B Protein

[0816] In this example size exclusion chromatography and ultrafiltrationwas used to purify recombinant BotB protein from the folding chaperonesand imidazole in IDA-purified material.

[0817] To enhance the solubility of the recombinant BotB protein duringscale-up, the protein was co-expressed with folding chaperones (see Ex.39). During the Ni-IDA purification step, the folding chaperonesco-eluted with the BotB protein in 800 mM imidazole; therefore, a secondpurification step was required to isolate the BotB free of foldingchaperones. Lane 3 of FIG. 35 contains proteins eluted from an IDAcolumn to which a lysate of pHisBotB kan lacIq T7/pACYCGro/BL21(DE3)cells had been applied; the proteins were resolved on a 4-15%polyacrylamide pre-cast gradient gel (Bio-Rad, Hercules, Calif.) rununder native conditions and then stained with Coomassie blue. In FIG.35, lanes 1 and 4 contain proteins present in peak 1 and peak 2 from aSephacryl S-100 column run as described below; lane 2 is blank.

[0818] As seen in lane 3 of FIG. 35, the IDA-purified sample consistsprimarily of the folding chaperones and the BotB protein. The fact thatthe chaperones and the Bot B antigen appear as two distinct bands undernative conditions suggested they were not complexed together andtherefore, it should be possible to separate them, using either agradient of imidazole concentrations or size exclusion methods.

[0819] In order to determine whether a gradient of imidazoleconcentrations could be used to separate the chaperone from the BotBprotein, a step gradient using imidazole at 200, 400, 600, and 800 mM in50 mM sodium phosphate, 0.5 M NaCl and 10% glycerol, pH 6.8 was appliedto an IDA column (containing proteins bound from a lysate of pHisBotBkan lacIq T7/pACYCGro/BL21(DE3) cells). By narrowing the range ofimidazole concentrations, it was hoped that the BotB and chaperoneproteins would differentially elute at different concentrations ofimidazole. Eluted proteins were monitored by absorbance at 280 mm andcollected either manually or with a fraction collector (BioRad). Proteinwas found to elute at 200 and 400 mM imidazole only.

[0820]FIG. 36 shows a Coomassie stained SDS-PAGE gel containing proteineluted during the imidazole step gradient. Lane 1 contains broad rangeMW markers (BioRad). Lane 2 contains BotB protein purified by IDAchromatography of an extract of pHisBotB/BL21(DE3) pLysS cells grown inshaker flask culture (i.e., no co-expression of chaperones; Ex. 35).Lane 3 contains a 20% w/v PEI clarified lysate of pHisBotB kan lacIqT7/pACYCGro/BL21(DE3) cells (i.e., the lysate prior to purification byIDA chromatography). Lanes 4 and 5 contain protein which eluted at 200or 400 mM imidazole, respectively. Lane 6 is blank. Lanes 7 and 8contain 1/5 the load present in lanes 4 and 5.

[0821] As shown in FIG. 36, both the chaperone and the BotB proteineluted in 200 mM imidazole, and more chaperone elutes in 400 μMimidazole, however no concentration of imidazole tested permitted theelution of BotB protein alone. Consequently, no significant purificationwas achieved using imidazole at these concentrations.

[0822] Because of the considerable difference in molecular weightsbetween the folding chaperone, which is a multimer with a totalmolecular weight around 400 kD (as determined on a Shodex KB 804 sizingcolumn by HPLC), and the recombinant BotB protein (molecular weightaround 50 kD), size exclusion chromatography was next examined for theability to separate these proteins.

[0823] a) Size Exclusion Chromatography

[0824] A column containing Sephacryl S-100 HR(S-100) (Pharmacia) waspoured (2.5 cm×24 cm; ˜110 ml bed volume). The column was equilibratedin a buffer consisting of phosphate buffered saline (10 mM potassiumphosphate, 150 mM NaCl, pH 7.2) and 10% glycerol (Mallinkrodt).Typically, 5 ml of the IDA-purified BotB protein was filtered through a0.45μ syringe filter and applied to the column, and the equilibrationbuffer was pumped through the column at a flow rate of 1 ml/minute.Eluted proteins were monitored by absorbance at 280 nm and collectedeither manually or with a fraction collector. Appropriate tubes werepooled, if necessary, and the protein was quantitated by absorbance at0.280 nm and/or by BCA protein assay. The isolated peaks were thenanalyzed by native and/or SDS-PAGE to identify the protein and evaluatethe purity.

[0825] Because of its larger size, the folding chaperone eluted first,followed by the recombinant BotB protein. A smaller third peak wasobserved which failed to stain when analyzed by SDS-PAGE and thereforewas presumed to be imidazole.

[0826] SDS-PAGE analysis (12.5% polyacrylamide, reduced samples) wasused to evaluate the purity of the IDA-purified recombinant BotB proteinbefore and after S-100 purification. The results are shown in FIG. 33.

[0827] In FIG. 33, lane 1 contains broad range MW markers (BioRad). Lane5 contains IDA-purified BotB protein. Lane 6 contains IDA-purified BotBprotein following S-100 purification. Lane 7 is blank (lanes 2-4 werediscussed in Ex. 34 above).

[0828] The results shown in FIG. 33 show that the IDA-purified BotB issignificantly contaminated with the folding chaperone (molecular weightabout 60 kD under reducing conditions; lane 6). Following S-100purification, the amount of folding chaperone present in the BotB samplewas reduced dramatically (lane 7). Visual inspection of the Coomassiestained SDS-PAGE gel revealed that after S-100 purification, >90% of thetotal protein present was BotB.

[0829] The IDA-purified BotB and the S-100-purified BotB samples wereanalyzed by HPLC on a size exclusion column (Shodex KB 804); thisanalysis revealed that the BotB protein represented 64% of the totalprotein in the IDA-purified sample and that following S-100purification, the BotB protein represented >95% of the total protein inthe sample.

[0830] The IDA-purified BotB material was also applied to a ACA 44(SpectraPor, Houston, Tex.) column. The ACA 44 resin is equivalent tothe S-100 resin and chromatography using the ACA 44 resin was carriedout exactly as described above for the S-100 resin. The ACA 44 resin wasfound to separate the recombinant BotB protein from the foldingchaperone. The ACA 44-purified BotB sample was analyzed for endotoxinusing the LAL assay (Associates of Cape Cod) as describe in Example 24.Two aliqouts of the ACA 44-purified BotB preparation were analyzed andwere found to contain either 58 to 116 EU/mg recombinant protein or 94to 189 EU/mg recombinant protein.

[0831] These results demonstrate that size exclusion chromatography canbe used to purify the recombinant BotB protein from the foldingchaperone and imidazole in IDA-purified material.

[0832] b) Ultrafiltration for the Separation of Recombinant BotB Proteinand Chaperones

[0833] Ultrafiltration was examined as an alternative method for theseparation recombinant BotB protein and folding chaperones inIDA-purified material. While in this example only mixtures of BotB andchaperones were separated by ultrafiltration, this technique is suitablefor use with recombinant BotA and BotE proteins as well provided thatthe wash buffers used are altered as necessary to take into accountdifferent requirements for solubility.

[0834] The recombinant BotB protein and folding chaperones wereseparated using a two-step sequential ultrafiltration method. The firstmembrane used had a nominal molecular weight cutoff (MWCO) ofapproximately 100 kD; this membrane retains the larger folding chaperonewhile allowing the smaller recombinant protein to pass through. Theaddition of several volumes of wash buffer may be required toefficiently wash the recombinant protein through the membrane. Thesecond step utilized a membrane with a nominal MWCO of approximately 10kD. During this step, the recombinant antigen was retained by themembrane and could be concentrated to the degree desired and theimidazole and excess wash buffer passed through the membrane.

[0835] Twenty-seven milliliters of an IDA-purified BotB preparation wasultrafiltered through a 47 mm YM 100 (100 kD MWCO) membrane (Amicon) ina 50 ml stirred cell (Amicon). The membrane was washed in dd H₂O priorto use as recommended by the manufacturer. Six volumes of 10% glycerolin PBS were washed through to remove most of the recombinant BotBprotein and this wash was collected in a separate vessel. The resultingBotB protein-rich filtrate was then concentrated 12-fold using a YM 10(10 kD MWCO) membrane (Amicon), to a final volume of 14 ml. The YM 100and YM 10 concentrates were analyzed along with the lysate startingmaterial by native PAGE using a 4-15% pre-cast gradient gel (BioRad).The results are shown in FIG. 37.

[0836] In FIG. 37, lane 1 contains IDA-purified BotB derived from ashaker flask culture (i.e., no co-expression of chaperones; Ex. 35);lane 2 contains a 20% w/v PEI clarified lysate of pHisBotB kan lacIqT7/pACYCGro/BL21(DE3) cells; lane 3 shows the lysate of lane 3 after IDApurification; lane 4 contains the YM 10 concentrate and lane 5 containsthe YM 100 concentrate.

[0837] The results shown in FIG. 37 demonstrate that the recombinantBotB protein can be purified away from the folding chaperone byultrafiltration through a 100 kD MWCO membrane (lane 4), leaving thechaperone protein in the 100 kD concentrate (lane 5). Analysis of thesample in lane 5 also showed that very little of the BotB protein wasretained by the 100 kD MWCO membrane after 6 volumes of wash buffer hadbeen applied.

[0838] The BotB samples following IDA chromatography and followingultrafiltration through the YM 100 membrane were anlyzed by HPLC on asize exclusion column (Shodex KB 804); this analysis revealed that theBotB protein represented 64% of the total protein in the IDA-purifiedsample and that following ultrafiltration through the YM 100 membrane,the BotB protein represented >96% of the total protein in the sample.

[0839] The BotB protein purified by ultrafiltration through the YM 100membrane was examined for endotoxin using the LAL assay (Associates ofCape Cod) as describe in Example 24. Two aliqouts of the YM 100-purifiedBotB preparation were analyzed and were found to contain either 18 to 36EU/mg recombinant protein or 125 to 250 EU/mg recombinant protein.

[0840] The above results demonstrate that size exclusion chromatographyand ultrafiltration can be used to purify recombinant botulinal toxinproteins away from folding chaperones.

Example 41 Cloning and Expression of the C Fragment of the C. botulinumSerotype E Toxin Gene

[0841] The C. botulinum type E neurotoxin gene has been cloned andsequenced from several different strains [Poulet et al. (1992) Biochem.Biophys. Res. Commun. 183:107 (strain Beluga); Whelan et al. (1992) Eur.J. Biochem. 204:657 (strain NCTC 11219); Fujii et al. (1990) Microbiol.Immunol. 34:1041 (partial sequence of strains Mashike, Iwani and Otaru)and Fujii et al. (1993) J. Gen. Microbiol. 139:79 (strain Mashike)]. Thenucleotide sequence of the type E toxin gene is available from the EMBLsequence data bank under accession numbers X62089 (strain Beluga) andX62683 (strain NCTC 11219). The nucleotide sequence of the coding region(strain Beluga) is listed in SEQ ID NO:49. The amino acid sequence ofthe C. botulinum type E neurotoxin derived from strain Belgua is listedin SEQ ID NO:50. The nucleotide sequence of the coding region (strainNCTC 11219) is listed in SEQ ID NO:51. The amino acid sequence of the C.botulinum type E neurotoxin derived from strain NCTC 11219 is listed inSEQ [D NO:52.

[0842] The DNA sequence encoding the native C. botulinum serotype E Cfragment gene derived from the Beluga strain can be expressed as ahistidine-tagged protein using the pETHisb vector; the resulting codingregion is listed in SEQ ID NO:53 and the corresponding amino acidsequence is listed in SEQ ID NO:54. The DNA sequence encoding the Cfragment of the native C. botulinum serotype E gene derived from theNCTC 11219 strain can be expressed as a histidine-tagged fusion proteinusing the pETHisb vector; the resulting coding region is listed in SEQID NO:55 and the corresponding amino acid sequence is listed in SEQ IDNO:56. The C fragment region from any strain of C. botulinum serotype Ecan be amplified and expressed using the approach illustrated belowusing the C fragment derived from C. botulinum type E 2231 strain (ATCC# 17786).

[0843] The type E neurotoxin gene is synthesized as a single polypeptidechain which may be converted to a double-chain form (i.e., a heavy chainand a light chain) by cleavage with trypsin; unlike the type Aneurotoxin, the type E neurotoxin exists essentially only in thesingle-chain form. The 50 kD carboxy-terminal portion of the heavy chainis referred to as the C fragment or the H_(C) domain. Expression of theC fragment of C. botulinum type E toxin in heterologous hosts (e.g., E.coli) has not been previously reported.

[0844] The native C fragment of the C. botulinum serotype E toxin (BotE)gene was cloned and inserted into expression vectors to facilitateexpression of the recombinant BotE protein in E. coli. This exampleinvolved PCR amplification of the gene, cloning, and construction ofexpression vectors.

[0845] The BotE serotype gene was isolated using PCR as described forthe BotA serotype gene in Example 28. The C. botulinum type E strain wasobtained from the American Type Culture Collection (ATCC #17786; strain2231). The following primer pair was used in the PCR amplification:5′-CGCCATGGCTCTTTCTTCTTAT ACAGATGAT-3′ (5′ primer, engineered NcoI siteunderlined) (SEQ ID NO:57) and 5′-GCAAGCTTTTATTTTTCTTGCCATCCATG-3′ (3′primer, engineered HindIII site underlined, native gene terminationcodon italicized) (SEQ ID NO:58). The PCR product was inserted intopCRscript as described in Example 28. The resulting pCRscript BotE clonewas confirmed by restriction digestion, as well as, by obtaining thesequence of approximately 300 bases located at the 5′ end of the Cfragment coding region using standard DNA sequencing methods. Theresulting BotE sequence was identical to that of the published C.botulinum type E toxin sequence [Whelan et al (1992), supra].

[0846] The NheI(filled)/HindIII fragment from a pCRscript BotErecombinant was cloned into pETHisb vector as described for BotA Cfragment in Example 28. The resulting construct was termed pHisBotE.pHisBotE expresses the BotE gene under the control of the T7 lacpromoter and the resulting protein contains an N-terminal 10×His-tagaffinity tag.

[0847] The pHisBotE expression construct was transformed into BL21(DE3)pLysS competent cells and 1 liter cultures were grown, induced andhis-tagged proteins were purified utilizing a NiNTA resin (eluted in lowpH elution buffer) as described in Example 28. Total, soluble andpurified proteins were resolved by SDS-PAGE and detected by Coomassiestaining. The results are shown in FIG. 38.

[0848] In FIG. 38, lane 1 contains broad range MW markers (BioRad); lane2 contains a total protein extract; lane 3 contains a soluble proteinextract; lane 4 contains proteins present in the flow through from theNiNTA column (this sample was not diluted prior to loading and thereforerepresents a load 5× that of the load applied for the total and solubleextracts in lanes 2 and 3); lane 5 contains proteins eluted from theNiNTA column; lane 6 contains protein eluted from a NiNTA column whichhad been stored at −20° C. for 1 year.

[0849] The pHisBotE protein was expressed at moderate levels (7mg/liter) as a totally soluble protein. The purified protein migrated asa single band of the predicted MW.

[0850] Western blot hybridization utilizing a chicken anti-C. botulinumserotype E toxoid primary antibody (generated by immunization of hens asdescribed in Example 3 using C. botulinum serotype E toxoid) was alsoperformed on the total, soluble and purified BotE proteins. Samples ofBotA and BotB C fragments were also included on the gels to facilitateMW and immunogenicity comparisons. Strong immunoreactivity was detectedusing the anti-C. botulinum type E toxoid antibody only with the BotEprotein.

[0851] These results demonstrate that the native BotE gene sequences canbe expressed as a soluble his-tagged protein in E. Coli and purified bymetal-chelation affinity chromatography.

Example 42 Generation of Neutralizing Antibodies Using the RecombinantpHisBotE Protein

[0852] The ability of the purified pHisBotE protein to generateneutralizing antibodies was examined. Nine BALBc mice were immunizedwith BotE protein (purified as described in Ex. 41) using Gerbu GMIDPadjuvant (CC Biotech). The low pH elution was mixed with Gerbu adjuvantand used to immunize mice. Each mouse received a subcutaneous injectionof 100 μl antigen/adjuvant mix (35 μg antigen+1 μg adjuvant) on day 0.Mice were subcutaneously boosted as above on day 14 and bled on day 28.Mice were subsequently boosted and bled on day 70.

[0853] Anti-C. botulinum serotype E toxoid titers were determined in day28 serum from individual mice from each group using the ELISA protocoloutlined in Example 29 with the exception that the plates were coatedwith C. botulinum serotype E toxoid, and the primary antibody was achicken anti-C. botulinum serotype E toxoid. Seroconversion [relative tocontrol mice immunized with the p6xHisBotA antigen (Ex. 29)] wasobserved with all 9 mice immunized with the purified pHisBotE protein.

[0854] The ability of the anti-BotE antibodies to neutralize native C.botulinum type E toxin was tested in a mouse-C. botulinum neutralizationmodel using pooled mouse serum (see Ex. 23b). The LD₅₀ of purified C.botulinum type E toxin complex (Dr. Eric Johnson, University ofWisconsin, Madison) was determined by a intraperitoneal (IP) method[Schantz and Kautler (1978), supra] using 18-22 g female ICR mice. Theamount of neutralizing antibodies present in the serum of the immunizedmice was determined using serum antibody titrations. The various serumdilutions (0.01 ml) were mixed with 5 LD₅₀ units of C. botulinum type Etoxin and the mixtures were injected IP into mice. The neutralizationswere performed in duplicate. The mice were then observed for signs ofbotulism for 4 days. Undiluted serum from day 28 did not protect, whileundiluted, {fraction (1/10)} diluted and {fraction (1/100)} diluted day70 serum protected (1005 of animals) while {fraction (1/1000)} dilutedday 70 serum did not. This corresponds to a neutralization titer of50-500 lU/ml.

[0855] These results demonstrate that seroconversion occurred andneutralizing antibodies were induced when the recombinant BotE proteinwas utilized as the immunogen.

Example 43 Construction of Vectors to Facilitate Expression ofHis-Tagged BotE Protein in Fermentation Cultures

[0856] A number of expression vectors were constructed to facilitate theexpression of recombinant BotE protein in large scale fermentationculture. These constructs varied as to the strength of the promoterutilized (T7 or T7lac) and the presence of repressor elements (lacIq) onthe plasmid. The resulting constructs varied in the level of expressionachieved and in plasmid stability which facilitated the selection of aoptimal expression system for fermentation scaleup. This exampleinvolved a) construction of BotE expression vectors and b) determinationof expression levels in small scale cultures.

[0857] a) Construction of BotE Expression Vectors

[0858] The BotE expression vectors created for fermentation culture wereengineered to utilize the kanamycin rather than the ampicillinresistance gene, and contained either the T7 or T7lac promoter, with orwithout the lacIq gene for the reasons outlined in Example 30.

[0859] In all cases, the protein expressed by the various expressionvectors is the pHisBotE protein described in Example 41, with the onlydifferences between clones being the alteration of various regulatoryelements. Using the designations outlined below, the pHisBotE clone (Ex.41) is equivalent to pHisBotE amp T7lac.

[0860] i) Construction of pHisBotE kan lacIq T7lac

[0861] pHisBotE kan lacIq T7lac was constructed by inserting theXbaI/HindIII fragment of pHisBotE which contains the BotE gene sequencesinto XbaI/HindIII-cleaved pET24a vector. Proper construction wasconfirmed by restriction digestion.

[0862] ii) Construction of pHisBotE kan T7

[0863] pHisBotE kan T7 was constructed by ligating the BotE-containingXbaI/SapI fragment of pHisBotE kan lacIqT7lac to the T7promoter-containing XbaI/SapI fragment of pET23a. Proper constructionwas confirmed by restriction digestion.

[0864] iii) Construction of pHisBotE kan lacIqT7

[0865] pHisBotE kan lacIqT7 was constructed by inserting theBglII/HindIII fragment from pHisBotE kan T7 which contains the BotE genesequences into BglII/HindIII-cleaved pET24 vector. Proper constructionwas confirmed by restriction digestion.

[0866] b) Determination of BotE Expression Levels in Small ScaleCultures

[0867] The three BotE kan expression vectors described above weretransformed into B121(DE3) competent cells and 50 ml (2XYT+40 μg/ml kan)cultures were grown and induced with ITPG as described in Example 28.Total and soluble protein extracts from before and after induction madeas described in Example 28. The total and soluble extracts were resolvedon a 12.5% SDS-PAGE gel, and his-tagged proteins were detected on aWestern blot utilizing the NiNTA-alkaline phosphatase conjugate asdescribed in Example 31(c)(iii). The results showed that all three BotEcell lines expressed his-tagged proteins of the predicted MW for theBotE protein upon induction. The results also demonstrated that the twoconstructs that contained the T7 promoter expressed the BotE proteinbefore induction, while the T7lac promoter construct did not. Uponinduction, the T7 promoter-containing constructs induced to higherlevels than the T71α-containing construct, with the pHisBotE kanlacIqT7/B121(DE3) cells accumulating the maximal levels of BotE protein.

Example 44 Expression and Purification of pHisBotE From FermentationCultures

[0868] Based on the small scale inductions performed in Example 43, thepHisBotE kan lacIq T7/B121(DE3) strain was selected for fermentationscaleup. This example involved the fermentation and purification ofrecombinant BotE C fragment protein.

[0869] A fermentation with the pHisBotE kan lacIq T7/B121(DE3) strainwas performed as described in Example 31. The fermentation culture wasinduced 2 hrs post start of the glucose feed with 4 gm IPTG (finalconcentration=1.6 mM). The OD₆₀₀ was 42 at time of induction, then 46.5,48, 53 and 54 at 1-4 hrs post induction. Viable colony counts decreasedfrom 0-4 hr induction [131, 4 (28), 7 (3), 7, 8; dilution 3 utilized 6μl of dilution 2 cells; bracketed colonies are microcolonies]. All(32/32) colonies scored at the time of induction retained the BotEplasmid (kan resistant) and no colonies at induction grew on IPTG+Kanplates (no mutations detected). These results were indicative of strongpromoter induction, since colony viability reduced after induction, andthe culture stopped growing during fermentation (stopped at 54OD₆₀₀/ml).

[0870] Total and soluble extracts were resolved on a 12.5% SDS-PAGE geland total protein was detected by staining with Coomassie blue. Theresults are shown in FIG. 39.

[0871] In FIG. 39, lane 1 contains total protein from a pHisBotA kan T7lac/B121(DE3) pLysS fermentation (Ex. 24). Lanes 2-9 contain extractsprepared from the above pHisBotE kan lacIq T7/B121(DE3) fermentation;lanes 2-4 contain total protein extracts prepared at 0, 1 and 2 hourspost-induction, respectively. Lane 5 contains a soluble protein extractprepared at 2 hours post-induction. Lanes 6 and 7 contain total andsoluble extracts prepared at 3 hours post-induction, respectively. Lanes8 and 9 contain total and soluble extracts prepared at 4 hourspost-induction, respectively. Lane 10 contains broad range MW markers(BioRad).

[0872] The results shown in FIG. 39 demonstrate that moderate levelinduction of totally soluble Bot E protein was observed, increasing from1 to 4 hrs post induction (no expression was detected in uninducedcells). From a 2 liter fermentation harvest a 155 gm (wet wt) cellpellet was obtained and used to make a PEI-clarified lysate (1 liter inCRB, pH 6.8). The lysate was applied to a large scale IDA column and 200mg of BotE protein, which was found to be greater than 95% pure (asjudged by visual inspection of a Coomassie stained SDS-PAGE gel), wasrecovered. This represents 2.5% of the total soluble cellular protein(assuming a PEI lysate having a concentration of 8 mg protein/ml) andcorresponds to a yield of 100 mg BotE protein/liter of fermentationculture.

[0873] The above results demonstrate that high levels of the recombinantBotE protein can be expressed and purified from fermentation cultures.

Example 45 Removal of Imidazole From Purified Recombinant BotE ProteinPreparations

[0874] The expression of recombinant BotE protein, unlike the BotA andBotB proteins, did not require the presence of folding chaperones tomaintain solubility during scale-up. A size exclusion chromatographystep was included however to remove the imidazole from the sample andexchange the IDA elution buffer for one consistent with the BotAantigen.

[0875] A Sephacryl S-100 HR(S-100; Pharmacia) column was poured (2.5cm×24 cm; bed volume ˜110 ml). Under these conditions, the BotE proteinshould be retained by the beads to a lesser degree than the smallerimidazole, therefore the BotE protein should elute from the columnbefore the imidazole. The column was equilibrated in a buffer consistingof 50 mM sodium phosphate, 0.5 M NaCl, and 10% glycerol (all reagentsfrom Mallinkrodt). Five milliliters of the IDA-purified BotE protein(Ex. 44) was filtered through a 0.45μ syringe filter and applied to theS-100 column, and equilibration buffer was pumped through the column ata flow rate of 1 ml/minute. Eluted proteins were monitored by absorbanceat 280 nm, and collected either manually or with a fraction collector.Appropriate tubes were pooled if necessary, and the protein wasquantitated by absorbance at 280 nm and/or BCA protein assay. Theisolated peaks were then analyzed by native and/or SDS-PAGE to identifythe protein(s) and evaluate the purity.

[0876]FIG. 40 provides a representative chromatogram showing thepurification of IDA-purified BotE on the S-100 column. Even thoughfolding chaperones were not over-expressed with this antigen, a smallamount of protein eluted at a time consistent with the foldingchaperones expressed with BotA and BotB proteins (Gro) (see the firstpeak). The second peak in the chromatogram contained the BotE protein,and the third peak was presumably imidazole. This presumed imidazolepeak was isolated in comparable levels in IDA-purified BotA and BotBprotein preparations as well.

[0877] These results demonstrate that size exclusion chromatography canbe used to remove imidazole and traces of contaminating high molecularweight proteins from IDA-purified BotE protein preparations.

[0878] The S-100-purified BotE protein was tested for endotoxincontamination using the LAL assay as described in Example 24. Thispreparation was found to contain 64 to 128 EU/mg recombinant protein andis therefore substantially free of endotoxin.

[0879] The S-100 purified BotE was mixed with purified preparations ofBOLA and BotB proteins and used to immunize mice; 5 μg of each Botprotein was used per immunization and alum was included as an adjuvant.After two immunizations with this trivalent vaccine, the immunized micewere challanged with C. botulinum toxin. The immunized mice containedneutralizing antibodies sufficient to neutralize between 100,000 to1,000,000 LD₅₀ of either toxin A or toxin B and between 1,000 to 10,000LD₅₀ of toxin E. The titer of neutralizing antibodies directed againsttoxin E would be expected to increase following subsequent boosts withthe vaccine. These results demonstrate that a trivalent vaccinecontaining recombinant BotA, BotB and BotE proteins provokesneutralizing antibodies.

Example 46 Expression of the C Fragment of the C. botulinum Serotype CToxin Gene and Generation of Neutralizing Antibodies

[0880] The C. botulinum type C1 neurotoxin gene has been cloned andsequenced [Kimura et al. (1990) Biochem. Biophys. Res. Comm. 171:1304].The nucleotide sequence of the toxin gene derived from the C. botulinumtype C strain C-Stockholm is available from the EMBL/GenBank sequencedata banks under the accession number D90210; the nucleotide sequence ofthe coding region is listed in SEQ ID NO:59. The amino acid sequence ofthe C. botulinum type C1 neurotoxin derived from this strain is listedin SEQ ID NO:60.

[0881] The DNA sequence encoding the native C. botulinum serotype C₁-Cfragment gene derived from the C-Stockholm strain can be expressed usingthe pETHisb vector; the resulting coding region is listed in SEQ IDNO:61 and the corresponding amino acid sequence is listed in SEQ IDNO:62. The C fragment region from any strain of C. botulinum serotype Ccan be amplified and expressed using the approach illustrated belowusing the C fragment derived from C. botulinum type C C-Stockholmstrain. Expression of the C fragment of C. botulinum type C1 toxin inheterologous hosts (e.g. E. coli) has not been previously reported.

[0882] The C fragment of the C. botulinum serotype C1 (BotC1) toxin geneis cloned using the protocols and conditions described in Example 28 forthe isolation of the native BotA gene. A number of C. botulinum serotypeC strains (expressing either or both C1 and C2 toxin) are available fromthe ATCC [e.g., 2220 (ATCC 17782), 2239 (ATCC 17783), 2223 (ATCC 17784;a type C-β strain; C-β strains produce C2 toxin), 662 (ATCC 17849; atype C-α strain; C-α strains produce mainly C1 toxin and a small amountof C2 toxin), 2021 (ATCC 17850; a type C-a strain) and VPI 3803 (ATCC25766)]. Alternatively, other type C strains may be employed for theisolation of sequences encoding the C fragment of C. botulinum serotypeC toxin.

[0883] The following primer pair is used to amplify the BotC gene:5′-CGCCATGGC TTTATTAAAAGATATAATTAATG-3′ [5′ primer, engineered NcoI siteunderlined (SEQ ID NO:63)] and 5′-GCAAGCTTTTATTCACTTACAGGTAC AAAACC-3′[3′ primer, engineered HindIII site underlined, native gene terminationcodon italicized (SEQ ID NO:64)]. Following PCR amplification, the PCRproduct is inserted into the pCRscript vector and then the 1.5 kbfragment is cloned into pETHisb vector as described for BotA C fragmentgene in Example 28. The resulting construct is termed pHisBotC. Properconstruction is confirmed by DNA sequencing of the BotC sequencescontained within pHisBotC.

[0884] pHisBotC expresses the BotC gene sequences under thetranscriptional control of the T7 lac promoter and the resulting proteincontains an N-terminal 10×His-tag affinity tag. The pHisBotC expressionconstruct is transformed into BL21(DE3) pLysS competent cells and 1liter cultures are grown, induced and his-tagged proteins are purifiedutilizing a NiNTA resin (eluted in 250 mM imidazole, 20% glycerol) asdescribed in Example 28. Total, soluble and purified proteins areresolved by SDS-PAGE and detected by Coomassie staining and Western blothybridization utilizing a Ni-NTA-alkaline phosphatase conjugate (Qiagen)which recognizes his-tagged proteins as described in Example 31(c)(iii).This analysis permits the determination of expression levels of thepHisBotC protein (i.e., number of mg/liter expressed as a solubleprotein). The purified BotC protein will migrate as a single band of thepredicted MW (i.e., ˜50 kD).

[0885] The level of expression of the pHisBotC protein may be modified(increased) by substitution of the T7 promoter for the T7lac promoter,or by inclusion of the lacIq gene on the expression plasmid, and plasmidexpressed in BL21(DE3) cell lines in fermentation cultures as describedin Example 30. If only very low levels (i e., less than 0.5%) of solublepHisBotC protein are expressed using the above expression systems, thepHisBotC construct may be co-expressed with pACYCGro construct asdescribed in Example 32. In this case, the recombinant BotC protein mayco-purify with the folding chaperones. The contaminating chaperones maybe removed as described in Example 34. Preparations of purified pHisBotCprotein are tested for endotoxin contamination using the LAL assay asdescribed in Example 24.

[0886] The purifed pHisBotC protein is used to generate neutralizingantibodies. BALBc mice are immunized with the BotC protein using GerbuGMDP adjuvant (CC Biotech) as described in Example 36. The ability ofthe anti-BotC antibodies to neutralize native C. botulinum type C toxinis demonstrated using the mouse-C. botulinum neutralization modeldescribed in Example 36.

Example 47 Expression of the C Fragment of the C. botulinum Serotype DToxin Gene and Generation of Neutralizing Antibodies

[0887] The C. botulinum type D neurotoxin gene has been cloned andsequenced (Sunagawa et al. (1992) J. Vet. Med. Sci. 54:905 and Binz etal. (1990) Nucleic Acids Res. 18:5556]. The nucleotide sequence of thetoxin gene derived from the CB16 strain is available from theEMBL/GenBank sequence data banks under the accession number S49407; thenucleotide sequence of the coding region is listed in SEQ ID NO:65. Theamino acid sequence of the C. botulinum type D neurotoxin derived fromthe CB16 strain is listed in SEQ ID NO:66.

[0888] The DNA sequence encoding the native C. botulinum serotype D Cfragment gene derived from a BotD expressing strain can be expressedusing the pETHisb vector; the resulting coding region is listed in SEQID NO:67 and the corresponding amino acid sequence is listed in SEQ IDNO:68. The C fragment region from any strain of C. botulinum serotype Dcan be amplified and expressed using the approach illustrated belowusing the C fragment derived from C. botulinum type D CB16 strain.Expression of the C fragment of C. botulinum type D toxin inheterologous hosts (e g. E. coli) has not been previously reported.

[0889] The C fragment of the C. botulinum serotype D (BotD) toxin geneis cloned using the protocols and conditions described in Example 28 forthe isolation of the native BotA gene. A number of C. botulinum type Dstrains are available from the ATCC [e.g., ATCC 9633, 2023 (ATCC 17851),and VPI 5995 (ATCC 27517)].

[0890] The following primer pair is used to amplify the BotD gene:5′-CGCCATGGC TTTATTAAAAGATATAATTAATG-3′ [5′ primer, engineered NcoI siteunderlined (SEQ ID NO:63)] and 5′-GCAAGCTTTTACTCTACCCATCCTGGATCCCT-3′[3′ primer, engineered HindIII site underlined, native gene terminationcodon italicized (SEQ ID NO:69)]. Following PCR amplification, the PCRproduct is inserted into the pCRscript vector and then the 1.5 kbfragment is cloned into pETHisb vector as described for BotA C fragmentgene in Example 28. The resulting construct is termed pHisBotD.

[0891] pHisBotD expresses the BotD gene sequences under thetranscriptional control of the T7 lac promoter and the resulting proteincontains an N-terminal 10×His-tag affinity tag. The pHisBotD expressionconstruct is transformed into BL21(DE3) pLysS competent cells and 1liter cultures are grown, induced and his-tagged proteins are purifiedutilizing a NiNTA resin as described in Example 28. Total, soluble andpurified proteins are resolved by SDS-PAGE and detected by Coomassiestaining and Western blot hybridization utilizing a Ni-NTA-alkalinephosphatase conjugate (Qiagen) which recognizes his-tagged proteins asdescribed in Example 31 (c)(iii). This analysis permits thedetermination of expression levels of the pHisBotD protein (i.e., numberof mg/liter expressed as a soluble protein). The purified BotD proteinwill migrate as a single band of the predicted MW (i.e., ˜50 kD).

[0892] The level of expression of the pHisBotD protein may be modified(increased) by substitution of the T7 promoter for the T7lac promoter,or by inclusion of the lacIq gene on the expression plasmid, and plasmidexpressed in BL21(DE3) cell lines in fermentation cultures as describedin Example 30. If only very low levels (i.e., less than about 0.5%) ofsoluble pHisBotD protein are expressed using the above expressionsystems, the pHisBotD construct may be co-expressed with pACYCGroconstruct as described in Example 32. In this case, the recombinant BotDprotein may co-purify with the folding chaperones. The contaminatingchaperones may be removed as described in Example 34. Preparations ofpurified pHisBotD protein are tested for endotoxin contamination usingthe LAL assay as described in Example 24.

[0893] The purifed pHisBotD protein is used to generate neutralizingantibodies. BALBc mice are immunized with the BotD protein using GerbuGMDP adjuvant (CC Biotech) as described in Example 36. The ability ofthe anti-BotD antibodies to neutralize native C. botulinum type D toxinis demonstrated using the mouse-C. botulinum neutralization modeldescribed in Example 36.

Example 48 Expression of the C Fragment of the C. botulinum Serotype FToxin Gene and Generation of Neutralizing Antibodies

[0894] The C. botulinum type F neurotoxin gene has been cloned andsequenced [East et al. (1992) FEMS Microbiol. Lett. 96:225]. Thenucleotide sequence of the toxin gene derived from the 202F strain (ATCC23387) is available from the EMBL/GenBank sequence data banks under theaccession number M92906; the nucleotide sequence of the coding region islisted in SEQ ID NO:70. The amino acid sequence of the C. botulinum typeF neurotoxin derived from the 202F strain is listed in SEQ ID NO:71.

[0895] The DNA sequence encoding the native C. botulinum serotype F Cfragment gene derived from the 202F strain can be expressed using thepETHisb vector; the resulting coding region is listed in SEQ ID NO:72and the corresponding amino acid sequence is listed in SEQ ID NO:73. TheC fragment region from any strain of C. botulinum serotype F can beamplified and expressed using the approach illustrated below using the Cfragment derived from C. botulinum type F 202F strain. Expression of theC fragment of C. botulinum type F toxin in heterologous hosts (e.g., E.coli) has not been previously reported.

[0896] The C fragment of the C. botulinum serotype F (BotF) toxin geneis cloned using the protocols and conditions described in Example 28 forthe isolation of the native BotA gene. The C. botulinum type F 202Fstrain is obtained from the American Type Culture Collection (ATCC23387). Alternatively, sequences encoding the BotF toxin may be isolatedfrom any BotF expressing strain [e.g., VPI 4404 (ATCC 25764), VPI 2382(ATCC 27321) and Langeland (ATCC 35415)].

[0897] The following primer pair is used to amplify the BotF gene:5′-CGCCATGGC TATTCTAATTATATATTTTAATAG-3′ [5′ primer, engineered NcoIsite underlined (SEQ ID NO:74)] and 5′-GCAAGCTTTCATTCTTTCCATCCATTCTC-3′[3′ primer, engineered HindIII site underlined, native gene terminationcodon italicized (SEQ ID NO:75)]. Following PCR amplification, the PCRproduct is inserted into the pCRscript vector and then the 1.5 kbfragment is cloned into pETHisb vector as described for BotA C fragmentgene in Example 28. The resulting construct is termed pHisBotF.

[0898] pHisBotF expresses the BotF gene sequences under thetranscriptional control of the T7 lac promoter and the resulting proteincontains an N-terminal 10×His-tag affinity tag. The pHisBotF expressionconstruct is transformed into BL21(DE3) pLysS competent cells and 1liter cultures are grown, induced and his-tagged proteins are purifiedutilizing a NiNTA resin as described in Example 28. Total, soluble andpurified proteins are resolved by SDS-PAGE and detected by Coomassiestaining and Western blot hybridization utilizing a Ni-NTA-alkalinephosphatase conjugate (Qiagen) which recognizes his-tagged proteins asdescribed in Example 31(c)(iii). This analysis permits the determinationof expression levels of the pHisBotF protein (i.e., number of mg/literexpressed as a soluble protein). The purified BotF protein will migrateas a single band of the predicted MW (i.e., ˜50 kD).

[0899] The level of expression of the pHisBotF protein may be modified(increased) by substitution of the T7 promoter for the T7lac promoter,or by inclusion of the lacIq gene on the expression plasmid, and plasmidexpressed in BL21(DE3) cell lines in fermentation cultures as describedin Example 30. If only very low levels (i.e., less than about 0.5%) ofsoluble pHisBotF protein are expressed using the above expressionsystems, the pHisBotF construct may be co-expressed with pACYCGroconstruct as described in Example 32. In this case, the recombinant BotFprotein may co-purify with the folding chaperones. The contaminatingchaperones may be removed as described in Example 34. Preparations ofpurified pHisBotF protein are tested for endotoxin contamination usingthe LAL assay as described in Example 24.

[0900] The purifed pHisBotF protein is used to generate neutralizingantibodies. BALBc mice are immunized with the BotF protein using GerbuGMDP adjuvant (CC Biotech) as described in Example 36. The ability ofthe anti-BotF antibodies to neutralize native C. botulinum type F toxinis demonstrated using the mouse-C. botulinum neutralization modeldescribed in Example 36.

Example 49 Expression of the C Fragment of the C. botulinum Serotype GToxin Gene and Generation of Neutralizing Antibodies

[0901] The C. botulinum type G neurotoxin gene has been cloned andsequenced [Campbell et al. (1993) Biochimica et Biophysica Acta 1216:487and Binz et al. (1990) Nucleic Acids Res. 18:5556]. The nucleotidesequence of the toxin gene derived from the 113/30 strain (NCFB 3012) isavailable from the EMBL/GenBank sequence data banks under the accessionnumber X74162; the nucleotide sequence of the coding region is listed inSEQ ID NO:76. The amino acid sequence of the C. botulinum type Gneurotoxin derived from this strain is listed in SEQ ID NO:77.

[0902] The DNA sequence encoding the native C. botulinum serotype G Cfragment gene derived from the 113/30 strain can be expressed using thepETHisb vector; the resulting coding region is listed in SEQ ID NO:78and the corresponding amino acid sequence is listed in SEQ ID NO:79. TheC fragment region from any strain of C. botulinum serotype G can beamplified and expressed using the approach illustrated below using the Cfragment derived from C. botulinum type G 113/30 strain. Expression ofthe C fragment of C. botulinum type G toxin in heterologous hosts (e.g.,E. coli) has not been previously reported.

[0903] The C fragment of the C. botulinum serotype G (BotG) toxin geneis cloned using the protocols and conditions described in Example 28 forthe isolation of the native BotA gene. The C. botulinum type G 113/30strain is obtained from the NCFB. The following primer pair is used toamplify the BotG gene: 5′-CGCCATGGCTGAC ACAATTTTTAATACA AGT-3′ [5′primer, engineered NcoI site underlined (SEQ ID NO:80)] and5′-GCCTCGAGTTATTCTGTCCATCCTTCATCCAC-3′ [3′ primer, engineered XhoI siteunderlined, native gene termination codon italicized (SEQ ID NO:81)].Following PCR amplification, the PCR product is inserted into thepCRscript vector and then the 1.5 kb fragment is cloned into pETHisbvector as described for BotA C fragment gene in Example 28 with theexception that the sequences encoding BotG are excised from thepCRscript vector by digestion with NcoI and AhoI and the NcoI site isblunted (the BotG sequences contain an internal HindIII site). ThisNcoI(filled)/XhoI fragment is then ligated to the pETHisb vector whichhas been digested with NheI and SalI and the NheI site is blunted. Theresulting construct is termed pHisBotG.

[0904] pHisBotG expresses the BotG gene sequences under thetranscriptional control of the T7 lac promoter and the resulting proteincontains an N-terminal 10×His-tag affinity tag. The pHisBotG expressionconstruct is transformed into BL21(DE3) pLysS competent cells and 1liter cultures are grown, induced and his-tagged proteins are purifiedutilizing a NiNTA resin as described in Example 28. Total, soluble andpurified proteins are resolved by SDS-PAGE and detected by Coomassiestaining and Western blot hybridization utilizing a Ni-NTA-alkalinephosphatase conjugate (Qiagen) which recognizes his-tagged proteins asdescribed in Example 31(c)(iii). This analysis permits the determinationof expression levels of the pHisBotG protein (i.e., number of mg/literexpressed as a soluble protein). The purified BotG protein will migrateas a single band of the predicted MW (i.e., ˜50 kD).

[0905] The level of expression of the pHisBotG protein may be modified(increased) by substitution of the T7 promoter for the T7lac promoter,or by inclusion of the lacIq gene on the expression plasmid, and plasmidexpressed in BL21(DE3) cell lines in fermentation cultures as describedin Example 30. If only very low levels (i.e., less than about 0.5%) ofsoluble pHisBotG protein are expressed using the above expressionsystems, the pHisBotG construct may be co-expressed with pACYCGroconstruct as described in Example 32. In this case, the recombinant BotGprotein may co-purify with the folding chaperones. The contaminatingchaperones may be removed as described in Example 34. Preparations ofpurified pHisBotG protein are tested for endotoxin contamination usingthe LAL assay as described in Example 24.

[0906] The purifed pHisBotG protein is used to generate neutralizingantibodies. BALBc mice are immunized with the BotG protein using GerbuGMDP adjuvant (CC Biotech) as described in Example 36. The ability ofthe anti-BotG antibodies to neutralize native C. botulinum type G toxinis demonstrated using the mouse-C. botulinum neutralization modeldescribed in Example 36.

Example 50 Expression of Recombinant Botulinal Toxin Proteins inEucaryotic Host Cells

[0907] Recombinant botulinal C fragment proteins may be expressed ineucaryotic host cells, such as yeast and insect cells.

[0908] a) Expression in Yeast

[0909] Botulinal C fragments derived from serotypes A, B, C, D, E, F andG may be expressed in yeast cells using a variety of commerciallyavailable vectors. For example, the pPIC3K and pPIC9K expression vectors(Invitrogen) may be employed for expression in the methylotrophic yeast,Pichia pastoris. When the pPIC3K vector is employed, expression of thebotulinal C fragment protein will be intracellular. When the pPIC3Kvector is employed, the botulinal C fragment protein will be secreted(the alpha factor secretion signal is provided on the pPIC9K vector).

[0910] DNA sequences encoding the desired C fragment is inserted intothese vectors using techniques known to the art. Briefly, the desiredbotulinal expression cassette (including sequences encoding the his-tag;described in the preceding examples) is amplified using the PCR inconjunction with primers that incorporate unique restriction sites atthe termini of the amplified fragment. Suitable restriction enzyme sitesinclude SnaBI, EcoRI, AvrII and NotI. When the botulinal C fragment isto be expressed using the pPIC3K vector, the initiator methionine (ATG)is provided by the desired Bot gene sequence and a Kozak consensussequence is engineered upstream of the ATG (e.g., ACCATGG).

[0911] The amplified restriction fragment containing the botulinal Cfragment gene is then cloned into the desired expression vector.Recombinant clones are integrated into the Pichia pastoris genome andrecombinant protein expression is induced using methanol following themanufacturer's instructions (Invitrogen Pichia expression kit manual).

[0912]C. botulinum genes are A/T rich and contain multiple sequencesthat are similar to yeast transcriptional termination signals (e.g.,TTTTTATA). If premature transcription termination is observed when thebotulinal C fragment genes are expressed in yeast, the transcriptiontermination signals present in the C fragment genes can be removed byeither site directed mutagenesis (utilizing the pALTER system; Promega)or by construction of synthetic genes utilizing overlapping syntheticprimers.

[0913] The botulinal C fragment genes may be expressed in other yeastcells using other commercially available vectors [e.g., using the pYES2vector (Invitrogen) and S. cerevisiae cells (Invitrogen)].

[0914] b) Expression in Insect Cells

[0915] Botulinal C fragments derived from serotypes A, B, C, D, E, F andG may be expressed in insect cells using a variety of commerciallyavailable vectors. For example, the pBlueBac4 transfer vector(Invitrogen) may be employed for expression in Spodoptera frugiperda(Sf9) insect cells (baculovirus expression system) (equivalentbaculovirus vectors and host cells are avaialble from other vendors,e.g., Pharmingen, San Diego, Calif.). Botulinal C fragments contained onNcoI/HindIII fragments contained within the pHisBotA-G expressionconstructs (described in the preceding examples) are cloned into thepBlueBac4 vector (digested with NcoI and HindIII); the NcoI site presenton the C fragment constructs overlaps with the start codon of the fusionproteins. In the case of botulinal C fragment clones that containinternal HindIII sites (e.g., using the BotG sequences described in Ex.49), the C fragment gene is contained within a NcoI/XhoI fragment on thepHisBot construct. This NcoI/XhoI fragment is excised from pHisBot andinserted into pBlueBac4 digested with NcoI and SalI. Recombinantbaculoviruses are made and the desired recombinant C fragment isexpressed in Sf9 cells using the protocols provided by the manufacturer(Invitrogen MaxBac manual). The resulting constructs will express thepHisBot protein intracellularly (including the N-terminal his-tag) underthe control of the polyhedrin promoter. For extracellular secretion ofbotulinal C fragment proteins, the C fragment sequences from the pHisBotconstructs are cloned into the pMelBacB vector (Invitrogen) as describedabove for the pBlueBac4 vector. When the pMelBacB vector is employed,the his-tagged botulinal C fragment proteins are secreted (utilizing avector-encoded honeybee melittin secretion signal) and contain a nineamino acid extension at the N-terminus.

[0916] His-tagged botulinal C fragments expressed in yeast or insectcells are purified using metal chelation columns as described in thepreceding examples.

[0917] From the above it is clear that the present invention providescompositions and methods for the preparation of effective multivalentvaccines against C. botulinum neurotoxin. It is also contemplated thatthe recombinant botulinal proteins be used for the production ofantitoxins. All publications and patents mentioned in the abovespecification are herein incorporated by reference. Variousmodifications and variations of the described method and system of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention.

1. A host cell containing a recombinant expression vector, said vector encoding a protein comprising at least a portion of a Clostridium botulinum toxin, said toxin selected from the group consisting of type B toxin and type E toxin.
 2. The host cell of claim 1, wherein and said host cell is capable of expressing said protein at a level greater than or equal to 5% of the total cellular protein.
 3. The host cell of claim 1, wherein and said host cell is capable of expressing said protein as a soluble protein at a level greater than or equal to 0.25% of the total soluble cellular protein.
 4. The host cell of claim 1, wherein said host cell is an Escherichia coli cell.
 5. The host cell of claim 1, wherein said host cell is an insect cell.
 6. The host cell of claim 1, wherein said host cell is a yeast cell.
 7. A host cell containing a recombinant expression vector, said vector encoding a fusion protein comprising a non-toxin protein sequence and at least a portion of a Clostridium botulinum toxin, said toxin selected from the group consisting of type B toxin and type E toxin.
 8. The host cell of claim 7, wherein said portion of said toxin comprises the receptor binding domain.
 9. The host cell of claim 7, wherein said non-toxin protein sequence comprises a poly-histidine tract.
 10. A vaccine comprising a fusion protein, said fusion protein comprising a non-toxin protein sequence and at least a portion of a Clostridium botulinum toxin, said toxin selected from the group consisting of type B toxin and type E toxin.
 11. The vaccine of claim 10 further comprising a fusion protein comprising a non-toxin protein sequence and at least a portion of Clostridium botulinum type A toxin.
 12. The vaccine of claim 10, wherein said portion of said Clostridium botulinum toxin comprises the receptor binding domain.
 13. The vaccine of claim 10 wherein said non-toxin protein sequence comprises a poly-histidine tract.
 14. The vaccine of claim 10, wherein said vaccine is substantially endotoxin-free.
 15. A method of generating antibody directed against a Clostridium botulinum toxin comprising: a) providing in any order: i) an antigen comprising a fusion protein comprising a non-toxin protein sequence and at least a portion of a Clostridium botulinum toxin, said toxin selected from the group consisting of type B toxin and type E toxin, and ii) a host; and b) immunizing said host with said antigen so as to generate an antibody.
 16. The method of claim 15, wherein said antigen further comprises a fusion protein comprising a non-toxin protein sequence and at least a portion of Clostridium botulinum type A toxin.
 17. The method of claim 15, wherein said portion of said Clostridium botulinum toxin comprises the receptor binding domain.
 18. The method of claim 15 wherein said non-toxin protein sequence comprises a poly-histidine tract.
 19. The method of claim 15 wherein said host is a mammal.
 20. The method of claim 19 wherein said mammal is a human.
 21. The method of claim 15 further comprising step c) collecting said antibodies from said host.
 22. The method of claim 21 further comprising step d) purifying said antibodies.
 23. The antibody raised according to the method of claim
 15. 24. The antibody raised according to the method of claim
 16. 